Journal of Ethnopharmacology 210 (2018) 198–208

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Phytochemical analysis, molecular docking and antiamnesic effects of MARK

methanolic extract of Silybum marianum (L.) Gaertn seeds in scopolamine
induced memory impairment in mice
Nausheen Nazira, Nasiara Karimb, Heba Abdel-Halimc, Imran Khand, Syed Fazal Wadooda,
⁎
Muhammad Nisara,
a
Department of Botany, University of Malakand, Chakdara, Dir (Lower), 18800 KP, Pakistan
b
Department of Pharmacy, University of Malakand, Chakdara, Dir (Lower), 18800 KP, Pakistan
c
Faculty of Pharmacy and Medical Sciences, University of Petra, Amman 11196, Jordan
d
Department of Pharmacy, University of Swabi, KP, Pakistan

A R T I C L E I N F O A BS T RAC T

Keywords: Ethnopharmacological relevance: Silybum marianum (L.) Gaertn and its main component silymarin have
Acetyl cholinesterase been extensively studied and have been found effective in various neurological disorders.
Induced fit docking Aims of the study: The aim of the current study is to identify phytoconstituents in the methanolic extract (Me.
Rutin Ext) of Silybum marianum (L.) Gaertn seeds and to study in-vivo the anti-amnesic effects along with in vitro
Morin
antioxidant and acetylcholinesterase (AChE) and buteryl cholinesterase (BChE) inhibition potential. Induced fir
Quercetin
Alzheimer's disease
docking (IFD) results have confirmed that quercetin, morin and rutin showed good affinity when docked into
AChE binding site.
Materials and methods: The present study investigates the in-vitro AChE and BChE inhibition potential of the
Me-Ext of Silybum marianum (L.) Gaertn at various concentrations (31.25, 62.50, 125, 250, 500, 1000 µg/mL)
using Ellman's spectrophotometric analysis, while antioxidant potential against DPPH and ABTS were
determined using Brand-Williams spectrophotometric method. Furthermore, the in-vivo anti-amnesic effects
of Me. Ext at the dose level of 50, 100 and 200 mg/kg were also evaluated using scopolamine -induced memory
impairment in mice in the novel object recognition test (NORT) and Y-maze test.
Results: The Me. Ext showed a concentration dependent inhibition of AChE and BChE with IC50 values of 110
and 130 µg/mL respectively and antioxidant activity against DPPH and ABTS with IC50 values 280 and 220 µg/
mL, respectively. In mice, Me. Ext reversed the amnesia induced by scopolamine as indicated by a dose-
dependent increase in spontaneous alternation performance in the Y-maze task (p < 0.05 versus scopolamine)
and increase in the discrimination index in the NORT comparable to the standard drug donepezil 2 mg/kg.
HPLC-UV analysis showed the presence of quercetin, rutin and morin. Induced fit docking (IFD) was performed
using quercetin, rutin and morin, Glide Gscore and IFD score of all compounds were consistent with their
experimental AChE inhibitory activities.
Conclusion: The results indicate that Silybum marianum (L.) Gaertn could be a new source for the isolation of
phytoconstituents useful in cognition and memory disorders such as Alzheimer's disease.

1. Introduction development of dementia. Hence, Alzheimer's disease and dementia

might be treated to reinstate the level of ACh by inhibiting cholinester-
During metabolic process free radicals are produced known as ase enzymes: AChE and BChE. Inhibition of cholinesterase enzymes
reactive oxygen species (ROS). These ROS exert oxidative stress and cause the development of cholinergic transmission and reduced
are involved in the development of degenerative disorders including amyloid aggregation (Candy et al., 1983; Loizzo et al., 2008). In
Alzheimer's disease. Furthermore, Alzheimer's disease is also asso- developed countries these diseases are handled through proper treat-
ciated with a decrease in neurotransmitter, the ACh which causes the ment but patients of the developing and underdeveloped countries

⁎
Corresponding author.
E-mail addresses: [email protected] (N. Nazir), [email protected] (N. Karim), [email protected] (H. Abdel-Halim),
[email protected] (I. Khan), [email protected] (S.F. Wadood), [email protected] (M. Nisar).

http://dx.doi.org/10.1016/j.jep.2017.08.026
Received 28 February 2017; Received in revised form 8 May 2017; Accepted 21 August 2017
Available online 24 August 2017
0378-8741/ © 2017 Elsevier B.V. All rights reserved.
N. Nazir et al. Journal of Ethnopharmacology 210 (2018) 198–208

mainly depends on traditional herbal medicine. Plants have significant as bedding, provided water and normal pellet diet and maintained
role as traditional herbal medicine for curing of various diseases under normal laboratory conditions such as temperature, humidity and
(Murad et al., 2013). Plants with antioxidant properties getting more 12 h light-dark cycle. All animal procedures have been approved by the
importance and more than 80% of the world's population living in the Departmental Animal Ethical Committee (DAEC/PHARM/2012/14)
developing countries primarily depends on herbal medicines for their and were conducted according to the UK animal scientific procedure
healthcare needs. According to ethno medicinal survey, 34% of the total act, 1986.
flora found in Pakistan has medicinal purposes (Shinwari, 2010). It has
been estimated that at least 6000 flowering plants were reported 2.3. Plant material
currently in Pakistan, among which 400–600 plants have medicinal
importance (Khan and Khan, 2007). According to literature survey it Silybum marianum (L.) Gaertn seeds were collected from different
has been estimated that up to 70,000 plant species are used medicinally areas of Malakand Division in the start of May; 2016. The plant
worldwide (Kumar et al., 2010). specimen was deposited with voucher number BGH.UOM.164 to the
Silybum marianum (L.) Gaertn is amongst the top selling herbs in herbarium, Department of Botany, University of Malakand, Pakistan.
the world. It is an annual and biennial plant that belongs to the
Compositae family and has been well-known since ancient times and 2.4. Extraction
suggested in traditional European and Asian medicine, mostly for
treatment of liver disorders (Biagi et al., 2016). The main active The extraction was carried out according to the method previously
constituent of this plant is silymarin, a light brownish yellow powder described (Radjadian et al., 2008). Extraction from Silybum marianum
consisting of seven flavonoid: taxifolin (TXF), silychristin (SCN), (L.) Gaertn seeds was a two step process: first the seeds (3 kg) were
silydianin (SDN), silybin A (SBNA), silybin B (SBNB), isosilybinin A defatted with 10 L n-hexane by maceration with shaking. The defatted
(ISBNA), and isosilybinin B (ISBNB) (Junqueira-Gonçalves et al., powder was then extracted with 10 L methanol at time periods of three
2015; Seal, 2016). The strong antioxidant activity and free radical days by maceration with shaking. Finally extracts were filtered and
scavenging ability of these flavonoids presented them as potential concentrated using rotary evaporator producing a yield of 8.3% w/w.
hepatoprotective, anti-inflammatory, antibacterial, antiallergic, anti-
mutagenic, antiviral, antineoplastic, and antithrombotic agents. It is 2.5. HPLC-UV Characterization
also shown to have vasodialatory action (Milic et al., 2013). The use of
Silybum marianum (L.) Gaertn has increased in Pakistan in recent For the preparation of extract sample for HPLC-UV characteriza-
years due to uncontrolled spread of hepatitis C virus and liver cirrhosis. tion, 20 mL of methanol and water (1:1 v/v) were mixed with 1 g of
Thus much attention has been paid to the liver protective effects of the powdered sample. This mixture was then heated on a water bath at
plant and its constituents. However, Silybum marianum (L.) Gaertn 50 °C for 1 h. After cooling, the sample was centrifuged at 4000 rpm for
seeds have also been traditionally used in the treatment of neurological 10 min. Then 2 mL from sample was filtered with Whatman filter paper
disorders including depression, Alzheimer's disease and epilepsy No. 1 into HPLC vials and labeled with proper codes.
(Borah et al., 2013) and studies have been mainly focused on the use The determination of phytoconstituents was carried out by means
of silymarin constituent in these disorders. Silymarin, is a complex of of Agilent 1260 infinity High-performance liquid chromatography
flavonolignans derived from the seeds of milk thistle (Silybum mar- (HPLC) system having basics parts like quaternary pump, auto-
ianum (L.) Gaertn) has been evaluated for its antiaging effects and has sampler, degasser and ultraviolet array detector (UVAD). The separa-
been found to extend the life span and reduce proteotoxicity in tion was achieved via Agilent Zorbax Eclipse XDB-C18 column. The
C.elgans model of Alzheimer's disease (Kumar et al., 2015). Other gradients system comprises of solvent B (methanol: acetic acid;
phytoconstituents found in Silybum marianum have not been evalu- deionized water, 100: 20: 180, v/v) and solvent C (methanol: acetic
ated for their usefulness in neurological disorders such as Alzheimer's acid: deionized water, 900: 20: 80, v/v) The efficient gradient program
disease. Thus, the present study aims to evaluate the memory enhan- was started with 100% B at 0 min, 85% B at 5 min, 50% B at 20 min,
cing effects of Me. Ext of Silybum marianum (L.) Gaertn and identify 30% B at 25 min, and 100% C from 30 to 40 min. After 25 min, elution
the other phytoconstiuents responsible for the anti-amnesic effects of occurred. For the analysis of the constituents, the ultraviolet array
the plant. detector (UVAD) was set to 280 nm and the spectra were recorded
from 190 to 500 nm. The identification was performed by using
2. Material and Methods retention times, available standards and UV spectra. Quantification
of the identified compounds was on the basis of % peak area (Zeb,
2.1. Chemicals 2015).

Acetyl cholin iodide (Sigma-Aldrich, UK), Butyryl cholin iodide 2.6. Anti-cholinesterase assays
(Sigma-Aldrich, Switzerland), 5,5-dithio-bis-nitrobenzoic acid (DTNB)
(Sigma-Aldrich, Germany), Electron eel AChE (type-VI-S) and Aquine In this assays, Me. Ext of the seeds of Silybum marianum (L.)
butyrly cholinesterase (Sigma-Aldrich, USA), DPPH (2, 20-diphenyl-1- Gaertn was analyzed spectrophotometrically for AChE and (BChE
picrylhydrazyl) from (Sigma-Aldrich, USA) and ABTS (2, 2′-azinobis-3- inhibition potential. Acetylcholine iodide and Buterylcholine iodide
ethylbenzothiazoline-6-sulfonic acid) were purchased from (Sigma- were used as substrates following Ellman's assay (Worek et al., 2012).
Aldrich, Germany). HPLC grade methanol from (Sigma-Aldrich, UK), In this assay 205 µL of plant samples dilution (31.05, 62.5, 125, 250,
donepezil and scopolamine were purchased from Sigma (St. Louis, MO, 500 and 1000 µg/mL) were added to a cuvette contained 5 µL of AChE
USA). All chemicals and solvents used were of analytical grades. (0.03 U/mL) and BChE (0.01 U/mL), 5 µL of DTNB catalyst kept in a
water bath having temperature 30 °C and then incubated for 15 min.
2.2. Animals After incubation, 5 µL of substrate were mixed to the mixture for
starting the reaction. Absorbance was measured at 412 nm for 4 min
A total number of 72 healthy Swiss male albino mice between 25 using double beam spectrophotometer. A yellow color 5-thio-2-nitro-
and 30 g were obtained from National Institute of Health Islamabad benzoate anion was formed by the reaction between thiocholines and
Pakistan, and maintained in the animal house of the Department of DTNB. Hydrolysis of substrate in the absence of enzyme was carried
Pharmacy, University of Malakand. The mice were housed in groups of out using white assay with no enzyme and plant samples. While the
six in individual cages made from stainless steel with soft wood pieces reaction mixture having all the above components without plant

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samples were used as a control. Percent enzymatic activity and percent The volume of oral (p.o.) and intraperitoneal (i.p.) administrations
enzymatic inhibition were calculated using the following formulas: was 1 mL/100 g b.w of mice. For each behavioral experiment per-
V = ∆Abs/∆t formed in this study, normal saline p.o., Me. Ext (50, 100 and 200 mg/
kg, p.o.), and donepezil (2 mg/kg, p.o.) were administered to mice 1 h
V before each trial and scopolamine (1 mg/kg, i.p.) was administered
%enzyme activity = ×100
V max 30 min before each trial.
%enzyme inhibition = 100 − %enzyme activity
(V show rate of reaction in the presence of inhibitor and Vmax show
the rate of reaction without inhibition). 2.10. Y-Maze Spontaneous Alternation Behavior

2.7. DPPH (2, 20-diphenyl-1-picrylhydrazyl) Scavenging assay Y-Maze Spontaneous Alternation Behavior was used to assess short
term memory (STM) function according to the method described by
DPPH free radical scavenging potential of Silybum marianum (L.) Hughes (2004). The Y-maze apparatus consisted of three arms of equal
Gaertn crude Me. Ext was determined by according to the method size, labeled as A, B, and C, respectively. Each arm was 20 cm long,
described by Brand-Williams and coworkers (Brand-Williams et al., 6 cm wide and 15.5 cm high and was oriented at an angle of 120° from
1995). DPPH solution were prepared by taking 24 mg in 100 mL of the other two. Mice were placed in one arm, and the sequence and the
methanol. The plant sample was prepared in methanol in the concen- number of arm entries were recorded via digital cameras and the
tration range of 1 mg/mL and diluted in the concentration range of entries were then calculated from the videos for each mouse over a five-
31.05, 62.5, 125, 250, 500 and 1000 µg/mL. 0.1 mL of Me. Ext minute session (e.g., CBABCC). Alternation was defined as consecutive
dilutions were mixed with 3.0 mL of DPPH solution and incubated at entries by a mouse into the three different arms. An arm entry was
23 °C for 30 min. Absorbance was calculated at 517 nm using UV- considered to be completed when the hind paws of the mouse were
spectrophotometer (Thermo electron corporation, USA). Ascorbic acid completely placed in an arm. The arena was cleaned using 70% v/v
used as a positive control. All results were taken in triplicates as mean ethanol between trials to avoid olfactory cues. The number of arm
± SEM. Percent radical scavenging activity was calculated using the entries, same arm returns (SAR), and alternate arm returns (AAR)
formula: were measured. Percentage of spontaneous alternation performance (%
SAP) was determined using the equation.
%Radical Scavenging = control absorbance −sample absorbance /control
absorbance×100 %SAP = [Actual alternations (total alternations)]/Possible alternations
(total number of arm entries−2 × 100.
2.8. ABTS (2, 2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) Free
Radical Scavenging Assay

Antioxidant activity of Silybum marianum (L.) Gaertn crude Me. 2.11. Novel object recognition test
Ext was carried out using ABTS (2, 2′-azinobis-3-ethylbenzothiazoline-
6-sulfonic acid) free radicals (Re et al., 1999). ABTS (7 mM) and The novel object recognition Test (NORT) was conducted according
Potassium per sulfate solution (2.45 mM) were prepared, mixed to the procedure described by Bengoetxea et al. (2015). NORT
thoroughly and kept in dark overnight for formation of free radicals. consisted of habituation, sample, and test phases. In the sample phase,
After incubation through addition of methanol (50%) absorption was each mouse was placed in an open field with two identical objects
adjusted in the range of 0.7 at 745 nm. Then 300 µL of plant extract (plastic ball) for five minutes. The mouse was then returned to its home
were mixed with 3.0 mL of ABTS solution in a cuvette. After 6 min cage. The apparatus was comprised of a white colored plywood box
absorbance was measured through double beam spectrophotometer. (40 cm × 40 cm × 66 cm) with a network floors that could be
Ascorbic acid was used as a positive control. Results were taken in effortlessly cleaned with 70%v/v ethanol after every trial. The appara-
triplicates. %ABTS scavenging activity was measured by using the tus was illuminated by a 60 W light suspended 50 cm over the crate.
following formula: The arena and objects were cleaned with 70% v/v ethanol between
trials to avoid olfactory cues. For STM, the test phase was performed
%ABTS Scavenging potential = control absorbance− sample five minutes after the sample phase. In the test phase, each mouse was
absorbance / control absorbance placed again in the open field in which one of the identical objects had
been replaced with a novel object (plastic square). The location of the
object was counterbalanced so that one half of the mice in each group
2.9. Experimental design
saw the novel object on the left side of the box arena, and the other half
saw the novel object on the right side of the box arena to eliminate bias
The mice were divided into six groups: and were given the following
of sides. A wash-out period of five days was allowed before the NORT
test solutions
was used to assess long-term memory in mice. The procedure was the
The mice were divided into six groups viz.
same as that for STM except that mice were presented in the test phase
24 h after the sample phase exposure. The time spent exploring each
Group I: Normal control, given normal saline 8 mL/kg, p.o.
object in each phase was recorded manually using a stopwatch. A
Group II: Negative control, given scopolamine 1 mg/kg, i.p.
mouse was scored as exploring when its head was oriented towards the
Group III: Positive control, given donepezil (2 mg/kg, p.o) +
object within a distance of 2 cm or when the nose was in contact with
scopolamine 1 mg/kg, i.p.
the object. Parameters measured included the time (in seconds) spent
Group IV: Treatment group, Me. Ext (50 mg/kg, p.o) + scopolamine
exploring familiar object (TF), time (in seconds) spent exploring the
1 mg/kg, i.p.
novel object (TN), and total time (in seconds) spent exploring both
Group V: Treatment group, Me. Ext,(100 mg/kg, p.o) + scopolamine
objects (TF+TN). Percentage of discrimination index) was determined
1 mg/kg, i.p.
using the following equation:
Group VI: Treatment group, Me. Ext (200 mg/kg, p.o) + scopola-
mine 1 mg/kg, i.p. DI(%) = TN/(TN+TF) × 100%

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2.12. Statistical analysis minimized using Macro Model module with the Merck Molecular Force
Field (MMFFs) (Schrödinger, LLC, New York, NY, 2016). Truncated
Data obtained from in vivo and in vitro experiments were Newton Conjugate Gradient (TNCG) minimization method was used
expressed as the mean ± standard error of mean (SEM) and plotted with 500 iterations and convergence threshold of 0.05 (kJ/mol)
with GraphPad Prism software (version 5.03) for graphical representa- (Schrödinger, LLC, New York, NY, 2016). IFD simulation was carried
tion. Statistical differences between the treatments and control groups out as per the consecutive steps in the IDF protocol; (Sherman et al.,
were evaluated using one-way analysis of variance (ANOVA) followed 2006) initially the ligands were docked into the rigid receptor model
by Dunnett's Multiple Comparison tests in GraphPad Prism (version with scaled-down van der Waals (vdW) radii, vdW scaling of 0.8 was
5.03). Two-way repeated measures (mixed model) ANOVA followed by used for both the protein the and ligand non-polar atoms. The Glide SP
Bonferroni posttests was used to compare the two objects in the object mode was used for the initial docking and 20 ligand poses were
recognition task in GraphPad Prism (version 5.03). Differences were retained for protein structural refinements (Halgren et al., 2004). The
considered significant if p value less than 0.05. docking site was set to be the centroid of the donepezil molecule from
the crystal structure. Secondly, Prime module was used to generate the
2.13. Induced fit docking induced-fit protein–ligand complexes; each of the 20 structures from
the previous step was subjected to side chain and backbone refine-
Induced fit docking (IFD) is a robust and accurate docking method ments. All amino acid residues with at least one atom within 5.0 Å
that is account for legend and receptor flexibility (Sherman et al., distance of each corresponding ligand pose were included in the Prime
2006). Molecular modeling and IFD simulation were performed using refinement (Jacobson et al., 2004). The ligand – receptor complexes
the Schrödinger® software suite (Schrödinger, LLC, New with minimum energy within 30 kcal/mol were then used in the final
York, NY, 2016). The atomic co-ordinates for the crystal structure of Glide docking and scoring step, each ligand was redocked into each
AChE complexed with donepezil was downloaded from the Protein refined low-energy receptor structure produced in the second step
Data Bank (PDB code: 1EVE, 2.5 Å resolution) (Kryger et al., 1998). using Glide XP (Friesner et al., 2006; Halgren et al., 2004). The IFD
The AChE structure was prepared with the Protein Preparation module score (kcal/mol) of: IFDScore = 1.0*Prime_Energy + 9.057*GlideScore
in the Schrödinger software. All crystal water molecules are kept as the + 1.428*Glide_Ecoul was calculated for each protein–ligand complex,
interactions between the ligand in the crystal structure are through this score accounts for both the protein–ligand interaction energy and
water molecules (Kryger et al., 1998). For protein preparation, the the total energy of the system. The more negative the IFD score, the
structure was subject to the following preparation steps: add hydro- more favorable the binding. The final ligand–protein complexes were
gens, assigning partial charges, and protonation states. Then the visualized and the different ligand-receptor interactions were studied.
protein structure was minimized using the OPLS force field in the
MacroModel module (Schrödinger, LLC, New York, NY, 2016).
Compounds (Fig. 1) were built by Maestro-molecule builder and energy

Fig. 1. Compounds used in induced fit docking calculations.

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Fig. 2. HPLC-UV chromatogram of Silybum marianum (L.) Gaertn Me.Ext.

3. Results respectively.

3.1. HPLC-UV based characterization of phenolic compounds 3.3. DPPH scavenging assay

HPLC is used to show good separation and resolution of all The antioxidant activity of Me. Ext of Silybum marianum against
antioxidants against their standard one. Silybum marianum (L.) DPPH free radicals showed a dose dependent response (Table 3). The
Gaertn seeds powder using selected protocol was run against standard Me. Ext showed 79.87 ± 0.23, 66.65 ± 0.30, 47.85 ± 0.17, 42.16 ± 0.14,
antioxidants quercetin, rutin and morin giving good separation of these 36.03 ± 0.31 and 30.21 ± 0.19% inhibition at the concentrations 1000,
phenolic compounds. These compounds were identified by comparing 500, 250, 125, 62.5 and 31.25 µg/mL with IC50 of 280 µg/mL
their peak position and retention time with that of standard phenolic (Table 3). The percent DPPH inhibition of Me. Ext was compared with
compounds. A typical HPLC-DAD chromatogram of Silybum maria- positive control (Ascorbic acid). Ascorbic acid displayed 89.08 ± 0.53%
num (L.) Gaertn is shown in Fig. 2. The peaks of quercetin, rutin and inhibition at 1000 µg/mL against DPPH with IC50 value of 60 µg/mL.
morin were recorded at 320 nm. The detailed identification of each
antioxidant with their respective peak position in chromatogram and
3.4. ABTS scavenging assay
retention time (Rt) is given in Table 1. Quercetin was eluted at
retention time of 10.4 min. morin was eluted at retention time of
The results of % ABTS inhibition potential of crude Me. Ext in ABTS
12.2 min and rutin, with retention time of 22.8 min. All these
free radical scavenging assay are shown in Table 3. The % inhibition of
antioxidants were identified from their respective standards.
Me. Ext were 72.57 ± 0.51, 67.76 ± 0.79, 51.72 ± 0.89, 44.11 ± 1.22,
Concentration of quercetin, rutin and morin were measured by
32.22 ± 0.52 and 13.47 ± 0.86 with their IC50 value 220 µg/mL at
comparing retention time and peak area of both sample and standards
concentrations 1000, 500, 250, 125, 62.5 and 31.25 µg/mL and
using the formula = Ax. Cs (µg/mL). V (mL)/As. Sample (gm)
displayed a dose dependent response. Percent ABTS inhibition of Me.
(Table 1). Cx= Concentration of unknown
Ext was compare with positive control (Ascorbic acid). Ascorbic acid
As=Peak area of standard has shown 88.14 ± 0.59 inhibitions at 1000 µg/mL against ABTS with
IC50 value 140 µg/mL.
Ax=Peak area of unknown

Cs=Concentration of standard (0.09 μg/mL) 3.5. Y-maze test

The results obtained in the Y maze test are shown in Fig. 1. The
3.2. Inhibition of AChE and BChE total number of arm entries were reduced in with scopolamine and
other treatment groups as compared to control, however this reduction
Table 2 shows the results of AChE and BChE inhibition of Me. Ext was not significant (p > 0.05). Furthermore, there was no significant
of Silybum marianum seeds and donepezil. The crude Me. Ext extract difference in the total number of arm entries among all groups
showed concentration dependent inhibition against AChE and BChE (Fig. 1a). The same arm returns were significantly increased in the
enzymes with IC50 values of 110 and 130 µg/mL respectively. Similarly scopolamine treated group compared to vehicle (p < 0.01). There was a
Donepezil was used as a standard drug and showed percent inhibition dose dependent reduction in the percentage of same arm returns for all
78.50 ± 0.45 against AChE and 88.30 ± 0.42 against BChE at highest treatment groups compared to scopolamine. There was no significant
concentration 1000 µg/mL with IC50 values of 95 and 80 µg/mL, difference in Me. Ext 100 and 200 mg/kg and donepezil 2 mg/kg.

Table 1
Identification and quantification of antioxidant constituents in seeds of Silybum marianum (L.) Gaertn.

Peak Retention time Antioxidant HPLC-DAD λ Peak Area of Peak Area of Concentration (µg/mL) Identification
(min) Identity max (nm) sample standard Reference

1 10.4 Quercetin 320 52.338 7089.285 0.007 Standard

2 12.2 Morin 320 43.752 2.017 0.002 Standard
3 22.8 Rutin 320 103.636 2241.216 0.021 Standard

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Table 2
AChE and BChE inhibition potential of Silybum marianum (L.) Gaertn seeds at various concentrations.

Samples Concentration (µg/mL) % AChE Mean ± S.E.M AChE IC50 (µg/mL) % BChE Mean ± S.E.M BChE IC50 (µg/mL)

Me. Ext 1000 77.20 ± 0.35 110 75.40 ± 0.52 130

500 75.25 ± 0.25 70.55 ± 0.33
250 62.25 ± 0.35 62.42 ± 0.32
125 53.58 ± 0.58 48.38 ± 0.61
62.5 39.47 ± 0.47 38.47 ± 0.50
31.25 35.28 ± 0.35 33.28 ± 0.36
Donepezil 1000 78.50 ± 0.45 95 88.30 ± 0.42 80
500 75.30 ± 0.35 75.55 ± 0.33
250 65.25 ± 0.37 68.72 ± 0.42
125 54.68 ± 0.68 59.48 ± 0.71
62.5 40.97 ± 0.57 45.37 ± 0.40
31.25 30.48 ± 0.35 31.29 ± 0.32

Abbreviations: AChE- Acetylcholin esterase; BChE- Buterylcholin esterase; Me. Ext- Methanolic Crude extract; Note: The data is represented as mean ± SEM, (n = 3).

Table 3
Percent DPPH and ABTS free radical scavenging activity of Silybum marianum (L.) Gaertn seeds at various concentrations.

Samples Concentration (µg/mL) % DPPH scavenging Mean ± S.E.M IC50 (µg/mL) % ABTS scavenging Mean ± S.E.M IC50 (µg/mL)

Me. Ext 1000 79.87 ± 0.23 280 72.57 ± 0.51 220

500 66.65 ± 0.30 67.76 ± 0.79
250 47.85 ± 0.17 51.72 ± 0.89
125 42.16 ± 0.14 44.11 ± 1.22
62.5 36.03 ± .031 32.22 ± 0.52
31.25 30.21 ± 0.19 13.47 ± 0.86
Ascorbic acid 1000 89.08 ± 0.53 60 88.14 ± 0.59 140
500 78.44 ± 0.48 77.22 ± 0.31
250 69.29 ± 0.49 68.11 ± 1.15
125 54.99 ± 0.99 51.89 ± 0.69
62.5 46.95 ± 0.22 47.58 ± 1.11
31.25 33.20 ± 1.03 41.27 ± 0.77

Abbreviations: DPPH; 2, 20-diphenyl-1-picrylhydrazyl, ABTS; 2, 2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid, Me. Ext Methanolic Crude extract; Note: The data is represented as
mean ± SEM, (n = 3).

Similarly, Me. Ext (100–200 mg/kg) caused a significant dose depen- Similar to the sample phase in the short term memory task, none of
dent increase in percentage of alternate arm returns compared to the groups showed a significant increase in the total time spent
scopolamine (p < 0.01). The % increase in alternate arm returns for Me. exploring the two objects in the long term task. Neither there was a
Ext 100 and 200 mg/kg was comparable to the standard drug donepezil significant increase in the time spent exploring each identical object
(2 mg/kg). The Me. Ext (100–200 mg/kg) also dose dependently among the groups (p > 0.05). In the test phase, there was a significant
increased the percentage of spontaneous alternation performance. increase in the time spent exploring the novel object with Me. Ext 200
The % increase in spontaneous alternation performance was significant and donepezil 2 mg/kg treated groups (p < 0.05). The preference to the
at 100 and 200 mg/kg of Me. Ext. The effect at 200 mg/kg of Me. Ext novel object in Me. Ext 200 mg/kg was comparable to the positive
was comparable to the standard drug donepezil (2 mg/kg) (Fig. 3). control group. Similarly, there was a significant increase in exploration
time of the familiar object with the Scop 1 mg/kg treated group (p <
0.05). There was a significant dose dependent increase in the percen-
3.6. Novel Object recognition test tage discrimination index (% DI) with Me. Ext 100, Me. Ext 200 mg/kg
and donepezil 2 mg/kg treated groups (p < 0.05; p < 0.01). The % DI
The results obtained with the novel object recognition test are with scopolamine 1 mg/kg was lower among all the groups and there
shown in Figs. 4 and 5. was a significant difference when it was compared with vehicle control.
In the sample phase of the short term memory task, there was no
significant difference in the total time spent exploring the two objects
(Fig. 2A). Similarly there was also no significant difference between the 3.7. Induced fit docking
extract treated groups and scopolamine treated group in the time spent
exploring each identical object. However, in the test phase, the group To validate the IFD process, the docked donepezil structure was
treated with Me. Ext (50, 100 and 200 mg/kg) + scopolamine (1 mg/ superimposed to donepezil obtained from the AChE crystal structure.
kg) and donepezil (2 mg/kg) and scopolamine (1 mg/kg) spent a longer RMSD value of 0.17 Å for all heavy atoms (excluding the hydrogen
time with the novel object than the identical one; but this difference in atoms) was observed (Fig. 6). Furthermore, the docked donepezil
activity was statistically not significant. In contrast the group treated receptor binding was found to be similar to those formed in the crystal
with scopolamine (1 mg/kg) spent significantly longer time with the structure (Kryger et al., 1998). Both in the crystal structure and docked
familiar object than the novel object (p < 0.05). The percentage donepezil the drug has shown to form the following interactions: the
discrimination index (%DI) was significantly higher for Me. Ext aromatic rings of donepezil are forming parallel π–π stacking with the
100 mg/kg + Scop (p < 0.05), Me. Ext 200+ scopolamine and donepezil Trp84 and Trp279 indole rings, a H-bond is found to be formed with
+ scopolamine (p < 0.01) treated groups compared to scopolamine the residues of the with Gly118, Gly119, Gly201, and Ser200. A
treated group. The % DI for all these groups was above 50%. The % DI cation–π interaction is believed to be formed between the protonated
with the scopolamine treated group was significantly lower when nitrogen of the piperidine ring and the phenyl ring of Phe330 (Fig. 6).
compared to the vehicle control (p < 0.01). IFD scores were found to be consistent with experimental AChE

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Fig. 3. Effect of Me.Ext (50, 100 and 200 mg/kg) of Silybum marianum (L.) Gaertn on the Y-maze task: (A) number of arm entries (B) Same arm returns (C) alternate arm returns (D)
spontaneous alternation performance. Values are mean ± SEM, n = 6. *p < 0.05, **p < 0.01 versus control and *p < 0.05, **p < 0.01 versus scopolamine (Scop) 1 mg/kg. (One way
ANOVA followed by Dunnett's posthoc test). DPZ (Donepezil).

inhibition activity (Khan et al., 2009; Szwajgier, 2013) as shown in including quercetin (Khan et al., 2009), morin (Remya et al., 2012) and
Table 4. Donepezil was found to have the most negative IFD score rutin (Cao et al., 2013). Thus docking studies were applied on these
while rutin had the lowest. In addition, quercetin and morin were compounds to validate the in-vitro and in-vivo findings of the current
found to occupy the same binding site but forming different interac- study.
tions (Fig. 7), which explains the less negative IFD score and hence the It is a well-established fact that cholinergic pathways are implicated
lower activity of the two compounds. However, rutin was shown inside in the pathophysiology of learning and memory disorders as these
the active site hypothetically but in reality it is not active against the pathways are projecting to the cerebral cortex and hippocampus
enzyme in in-vitro assay. This drastic difference between in-silico (Ferreira-Vieira et al., 2016; Muir, 1997). Studies revealed that
binding affinity and computational predictions was the fact that cholinergic deficits have shown a wide range of cognitive debilitations
molecular size of rutin is slightly larger than the opening slit on surface in Alzheimer's disease. Scopolamine, a cholinergic receptor antagonist
of AChE leading to the active site (Fig. 8). So practically, rutin can’t has widely been used to induce impairment in learning and memory via
penetrate the aromatic gorge of AChE to exert its inhibitory activity. blockade of central M1 muscarinic receptors. Scopolamine-induced
amnesia is the classical model to study the effects of nootropic agents in
learning and memory disorders including Alzheimer's disease
4. Discussion
(Blokland, 1995). Cholinomimetic agents have been shown to reverse
the amnesic effects of scopolamine in both animals and humans
In the present study the memory enhancing effect of Me. Ext of
(Budzynska et al., 2015). Thus AChE inhibitors which improve access
Silybum marianum (L.) Gaertn were investigated using in-vitro
to ACh in the synaptic cleft reverse the cognitive deficits induced by
cholineesterase's inhibition studies along with antioxidant potential.
scopolamine.
The cholinesterase's inhibitory effects of Me. Ext of Silybum marianum
Spontaneous alternation is the measure of exploration behavior in
(L.) Gaertn seeds were further complemented with in-vivo studies for
mice and is a reliable screening model to test the anti-amnesic effects of
possible nootropic and cognition enhancement effects. The Me. Ext of
pharmacological substances including natural products in scopola-
Silybum marianum (L.) Gaertn showed antioxidant activities as
mine-induced memory impairments (Sarter et al., 1988). Mice with
reported in previous studies (Anthony et al., 2013; Surai, 2015). The
impaired short term memory cannot recall which arm they had just
phytochemical investigation of Me. Ext of Silybum marianum (L.)
visited and thus show reduction in spontaneous alternation. In this
Gaertn using HPLC-UV profiling showed the presence of flavonoids

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Fig. 4. Effect of Me.Ext (50, 100 and 200 mg/kg) of Silybum marianum (L.) Gaertn in short-term memory NORT. (A) Exploration time in the sample phase ( B) Exploration time in the
test phase (C) Discrimination index. *p < 0.01 versus control and *p < 0.05, **p < 0.01 versus Scop 1 mg/kg. A and B were analyzed using Two-way repeated (Mixed model) ANOVA
followed by Bonferroni post test and C was analyzed by One-way ANOVA followed by Dunnett's posthoc test.

study we used Y-maze test for the evaluation of spontaneous alterna- In the sample phase of short term memory test, Me. Ext did not
tion behavior to assess working memory. cause a significant increase in the exploration time of each identical
In the Y-maze test, administration of scopolamine (1 mg/kg) object. Neither was there a significant difference in the total time spent
caused significant reduction in spontaneous alternation performance exploring the two objects between all scopolamine treated groups. This
in the Y maze test indicating short term memory impairment in mice. result suggests the absence of visual effects including blurred vision,
The Me. Ext of Silybum marianum (L.) Gaertn seeds reversed the loss of accommodation and dilation of pupil associated with scopola-
scopolamine induced-reduction in spontaneous alternation behavior mine administration at the dose level of 1 mg/kg.
and caused a significant increase in spontaneous alternation in mice at In contrast the group treated with Me. Ext (200 mg/kg) demon-
the dose level of 100 and 200 mg/kg. This indicates nootropic effect of strated clear preference for the novel object in the test phase whereas
Silybum marianum (L.) Gaertn extract in scopolamine induced scopolamine 1 mg/kg failed the novelty preference test indicating that
amnesia. Donezepil is used as memory enhances and it works by scopolamine causes short term memory impairment in mice. The
inhibiting AChE and thus increases ACh level. Donezepil mg/kg also antiamnesic effects of Me. Ext (200 mg/kg) were comparable to the
caused a significant increase in spontaneous alternation performance standard drug donezepil (2 mg/kg).
decreased by scopolamine. The increase in spontaneous alternation Similar to the short term memory task, there was no significant
behavior by donezepil 2 mg/kg was comparable to the Silybum difference among various groups in the sample phase of long term
marianum (L.) Gaertn Me. Ext (200 mg/kg), suggesting a similar memory task. However in the test phase Me. Ext 200 mg/kg caused a
mechanism for the extract. Furthermore, a reduction in same arm significant increase in the time spent exploring the novel object
returns and an increase in alternative arm entries by Me. Ext of compared to the familiar object. The exploration time of the novel
Silybum marianum (L.) Gaertn indicate anti-amnesic effects in cogni- object observed with Me. Ext 200 mg/kg was comparable to the
tion deficits. standard drug donezepil (2 mg/kg). Similar effects were observed in
The novel object recognition test is widely used animal model for the discrimination index. The % DI was significantly higher with Me.
the assessment of short and long term memories. In this study NORT Ext 200 mg/kg and donezepil 2 mg/kg indicating the ability of the
was used to assess the anti-amnesic effects of Me. Ext of Silybum animals to retain preference for the novel object in the long term
marianum (L.) Gaertn in scopolamine induced memory impairment. memory task at these doses.

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N. Nazir et al. Journal of Ethnopharmacology 210 (2018) 198–208

Fig. 5. Effect of Me.Ext (50, 100 and 200 mg/kg) of Silybum marianum (L.) Gaertn in long term memory NORT. (A) Exploration time in the sample phase (B) Exploration time in the
test phase (C) Discrimination index. *p < 0.05; *p < 0.01 versus control and *p < 0.05, **p < 0.01 versus Scop 1 mg/kg. A and B were analyzed by Two-way repeated (Mixed model)
ANOVA followed by Bonferroni post test and C was analyzed by One-way ANOVA followed by Dunnett's posthoc test.

Table 4
IFD Scores and IC50 values of donepezil and AChE inhibitors present in Silybum
marianum (L.) Gaertn seeds.

S.NO Compounds IC50 (µg/mL) IFD

Score

1 Donepezil 31.83 ± 0.49 (Moniruzzaman et al., 2015) −1147

2 Quercetin 95.8 (Ding et al., 2013) −1142
3 Morin 210 (Remya et al., 2012) −1134
4 Rutin No Sig Inhibition (Khan et al., 2009) −1112

BChE observed in this study. These enzymes are associated with the
degradation of ACh. Thus, inhibiting these enzymes causes an increase
in the level of ACh. The effects were comparable to the standard AChE
inhibitor; donezepil suggesting that the mechanism (s) of action
Fig. 6. The binding sites of AChE (1EVE) with Donepezil: native Donepezil (purple involved in Silybum marianum (L.) Gaertn may be similar to mechan-
carbons), IFD-generated Donepezil model (blue carbons) (For interpretation of the ism of action of donezepil.
references to color in this figure legend, the reader is referred to the web version of this
HPLC-UV analysis was also used for the quantification and resolu-
article.).
tion of different phenolic compounds. The phenolic constituents of the
Silybum marianum (L.) Gaertn Me. Ext were identified to be quercetin,
The antiamnesic effects of Me. Ext of Silybum marianum (L.)
morin and rutin. These compounds have been shown to inhibit AChE
Gaertn seeds in both the short term and long term memory task of the
in vitro (Khan et al., 2009; Szwajgier, 2013). IFD results confirmed the
novel object recognition test may be due to inhibition of the AChE and

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N. Nazir et al. Journal of Ethnopharmacology 210 (2018) 198–208

Fig. 7. Mode of binding of different compounds in the active site of AChE (1EVE). Hydrogen bonds are shown by dotted purple lines and π–π stacking as dotted blue line. (a) Donepezil,
(b) quercetin, (c) morin.

Fig. 8. Electrostatic and steric map of the acetylcholinesterase protein surface. In left figure, binding mode of the rutin docked inside the active of rutin calculated theoretically. In
middle figure, the closer view of rutin docked inside the active site is shown, only flavone ring is visible from the terminal slit at the surface of protein, which clearly shows the
inappropriate the size of the molecule to passed through the opening of aromatic gorge. In right figure, rutin is docked in the opening slit of rutin, which clearly explained the negative in-
vitro activity of the enzyme due to failure to pass through inlet on protein surface leading to the active sight of enzyme.

AChE inhibitory effect of quercetin and morin and showed good affinity manuscript for publication.
when docked into the AChE binding site. Glide Gscore and IFD score of
all compounds were consistent with their experimental activities. Thus Acknowledgement
the antiamnesic effects of Me. Ext of Silybum marianum (L.) Gaertn
observed in this study may be due to quercetin and morin constituents Dr Nasiara Karim acknowledges funding (Start- up research grant;
of the extract. The present study provides a considerable source of SRGP 935) from the Higher education commission of Pakistan for the
secondary metabolites which play a role as antioxidant and choline completion of this research work. The funding body did not contribute
esterase inhibitors. to the design or any other part of the research work.
The authors are also grateful to Prof. Mehboob ur Rahman, Post
5. Conclusion Graduate College Matta, Swat, Khyber Pakhtunkhwa, Pakistan for the
identification of plant.
In conclusion, Me. Ext of Silybum marianum (L.) Gaertn possesses
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