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Vol. 13(11), pp. 252-268, 10 June, 2019
DOI: 10.5897/JMPR2018.6728
Article Number: 3FF026261151
ISSN 1996-0875
Copyright © 2019
Author(s) retain the copyright of this article
http://www.academicjournals.org/JMPR
Journal of Medicinal Plants Research
Full Length Research Paper
Chemical fingerprint of Bacopa monnieri L. and
Rosmarinus officinalis L. and their neuroprotective
activity against Alzheimer's disease in rat model’s
putative modulation via cholinergic and
monoaminergic pathways
Nermien E. Waly1, Nora M. Aborehab2* and Mahitab H. El Bishbishy3
1Department of Physiology, Faculty of Medicine, Helwan University, Cairo, 11795, Egypt.
2Department of Biochemistry, Faculty of Pharmacy, MSA University, Giza 11787, Egypt.
3Department of Pharmacognosy, Faculty of Pharmacy, MSA University, Giza 11787, Egypt.
Received 26 December, 2018; Accepted 30 January, 2019
Alzheimer’s disease is characterized by progressive degeneration of cortical and hippocampal neurons.
This study aims to characterize the metabolic profiles of the hydro-ethanolic extracts of Bacopa
monnieri L. (BM) and Rosmarinus officinalis L. (RO) cultivated in Egypt via UPLC–ESI/MS analyses and
reveal their possible mechanism of the prophylactic effect(s) on neuro-degeneration in rat model of
Alzheimer’s disease (AD). Here, UPLC–ESI/MS analyses were employed for the characterization of
hydro-ethanolic extracts. Forty-two male albino rats were intra-peritoneally injected with Aluminum
chloride at a dose of 4.2 mg/kg to induce AD. The extracts of BM and RO were separately orally
administered at doses of 300 and 450 mg/kg, and Donazil® was orally administered at dose 2.5 mg/kg.
Serum levels of malondialdehyde (MDA), and total antioxidant capacity (TAC) were measured using
ELISA. Further, Amyloid β-protein, acetylcholinesterase (AChE), τ-protein and serotonin levels were
measured in brain tissue using ELISA. The UPLC–ESI/MS analyses revealed the presence of fifteen and
seventeen active metabolites in BM and RO extracts respectively which may account for their effects on
neuro-degeneration. Serum level of MDA, amyloid β-protein, AChE and τ-protein were significantly
decreased in herbal treated groups when compared to AD group (P value < 0.0001). On the other hand,
TAC and serotonin levels were significantly elevated in groups treated with BM and RO compared to AD
group (P value < 0.0001). Consequently, BM and RO extracts were found to have a potential
neuroprotective effect in AD rat model due to their variety of active metabolites.
Key words: Alzheimer's, serotonin, anti-AChE, antioxidant, Bacopa monnieri, Rosmarinus officinalis.
INTRODUCTION
Aluminum (Al) is the most abundant metal on earth, it can
enter the body through diet, drinking water, aluminum containing drugs and so enters the brain; deposited in the
cortex, hippocampus and cerebellum which are crucial for
*Corresponding author. E-mail: naborehab@msa.eun.eg.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution
License 4.0 International License
memory and cognition. Al was reported as a main risk
factor for the cause and development of neuro-
degenerative diseases as: Alzheimer’s disease (AD),
amyotrophic lateral sclerosis and Parkinson’s disease
(PD) (Thenmozhi et al., 2016).
Aluminum induced neurotoxicity was previously
reported by many authors in which administration of
aluminum chloride hexahydrate 25 mg/kg/day for one
month orally (Zaky et al. 2013) and daily treatment with
AlCl3 at dose 100 mg/kg orally for 42 days was observed
(Lin et al., 2015).
Alzheimer’s disease is characterized by progressive
degeneration of cortical and hippocampal neurons. This
results in deterioration of the persons’ memory and
cognitive ability (Ghoneim et al., 2015). AD is the most
common cause of dementia among elderly population
(Zhang et al., 2016) and has a progressive and
devastating nature that poses a huge financial and social
burden on families and caregivers of the elderly (Sica,
2015; Goren et al., 2016). Precise worldwide prevalence
of AD is difficult to estimate; however, prevalence of AD
is expected to increase in the coming decades as a
consequence of aging of the world population (Goren et
al., 2016; Brookmeyer et al., 2007; Carter, 2008).
Although not well understood, it has been postulated
that loss of cholinergic function at the central nervous
system as well as accumulation of reactive oxygen
species are the key components implicated in AD
pathophysiology (Bartus, 2000; Murphy and
Steenbergen, 2008). Accumulation of amyloid β (Aβ)
protein and appearance of neurofibrillary tangles of tau
(τ) protein are the most prominent pathological hallmarks
of AD (Iqbal and Grundke-Iqbal, 2010). To date, there is
no effective treatment for AD that is why finding
preventive measure to reduce the disease incidence is of
crucial significance (Sica, 2015).
Bacopa monnieri L. (BM), commonly known as
“Brahmi” named after Brahma, the creator god of the
Hindu pantheon of deities, is an herb that is used in
traditional Indian medicine for its antioxidant and anti-
inflammatory as well as memory-enhancing effect. This
effect is believed to date back more than 3000 years in
India (Rathee et al., 2008; Chaudhari et al., 2017).
Rosmarinus officinalis L. (RO) is used since antiquity to
enhance the memory. The ancient Egyptians had a
famous tradition of laying rosemary across the coffin or
upon a tombstone during the embalming process and it
was considered sacred to ancient Egyptians, Romans
and Greeks (Burlando, 2010). It was traditionally burned
for the Greek students prior to their exams to boost their
mental performances and was considered a loyalty
symbol between lovers due to this trait (Rathee et al.,
2008). Rosemary is an herb reportedly known for its
antioxidant and anti-inflammatory effects (Aruoma et al.,
1996; Lipton et al., 2016; Posadas et al., 2009).
Herbal extracts pose a potential hope for prevention
and treatment of many age-linked diseases such as
atherosclerosis (Kabiri et al., 2012), hypertension (Reinhart
Waly et al. 253
et al., 2008), type 2 Diabetes mellitus (Li et al., 2004) and
osteoarthritis (Aborehab et al., 2017). Focusing on
nootropic herbal extracts, it is well established that
Ginkgo biloba (Birks and Grimley, 2007), Piper nigrum
(Subedee et al., 2015), Hericium erinaceus (Zhang et al.,
2016), Withania somnifera (Bhattacharya et al., 2001)
and Panax ginseng (Petkov et al., 1993) extracts improve
learning and memory deficits and relieve the
neuropsychological symptoms associated with animal
models of AD. Recently, BM showed cholinergic effects
in mice similar to current treatments of AD (Le et al.,
2013). Also, attention has been drawn to use RO in
treatment of AD as its metabolite, carnosic acid (CA),
was found to enhance memory and learning of AD mice
model (Lipton et al., 2016).
The objective of this study is to characterize the
metabolic profiles of the hydro-ethanolic extracts of BM
and RO cultivated in Egypt and to investigate their
neuroprotective effects compared to standard medication
Donazil® as a selective acetylcholinesterase inhibitor as
well its potential mechanism of its neuroprotective actions
in a rat model of AD.
METHODOLOGY
Plant material
The aerial parts of “Brahmi” B. monnieri L. (Plantaginaceae) and
“Rosemary” R. officinalis L. (Lamiaceae) were provided from El
Orman Botanical Garden, Giza, Egypt and kindly identified by Dr.
Mohamed El-Gebaly (National Research Institute, Dokki, Giza,
Egypt). Voucher specimens of both collected samples were kept at
Pharmacognosy Department, Faculty of Pharmacy, October
University for Modern Sciences and Arts (MSA) with codes (MSA-
2017-8 and MSA-2017-9, respectively). The Fresh aerial parts (500
g) were air dried at room temperature, then ground into fine
powder. The extracts were obtained using a Soxhlet extraction over
a period of 8 h using 70% ethanol, filtered, and the solvent was
evaporated under reduced pressure in a rotatory evaporator at a
temperature as low as 45°C. The dried extracts were kept in a
desiccator for further chemical and biological investigations.
UPLC-Electro spray ionization-mass spectroscopy (UPLC–ESI-
MS) apparatus
40 µg of BM and RO hydro-ethanolic extracts were separately
dissolved in 1 mL HPLC grade methanol, filtered using a 0.2 μm
membrane disc filter and degassed by sonication prior to injection.
UPLC–ESI-MS analyses were performed on an Agilent® 1100
Series using ACQUITY UPLC - BEH C18 column, (1.7 µm - 2.1 ×
50 mm, i.d.), with an integrated pre-column. 10 μL of each extract
was eluted using gradient mobile phase composed of two eluents:
eluent A is nano-pure H2O acidified with 0.1% formic acid and
eluent B is MeOH acidified with 0.1% formic acid with the flow rate
of 0.2 mL/min for 35 min. A XEVO TQD triple quadruple instrument,
Waters® Corporation, Milford, MA01757, U.S.A mass spectrometer
connected to a PDA detector with standard flow cell (10 mm path
length, 14 µL volume, 40 bar maximum pressure) was used for
mass spectrometric analysis. ESI interface was employed in both
negative and positive ion modes using N2 as a drying and
nebulizing gas. At 250°C capillary temperature, the spray and
capillary voltages were 4.48 kV and 39.6 V respectively and the full
scan mode was in mass range of m/z 100–2000. The peaks and
254 J. Med. Plants Res.
spectra were interpreted using the Masslynx 4.1® software and
tentatively assigned by comparing their mass spectrums with the
reported data.
Chemicals and drugs
Aluminum chloride (AlCl3) was purchased from Sigma-Aldrich
Chemicals Co, Egypt, dissolved in saline 0.9% and injected
intraperitonealy (I.P) at dose 4.2 mg/kg/day for 28 days according
to methodology of Bitra et al. (2014) and Nayak and Chatterjee
(2001). Ethanol (analytical grade) was purchased from El
Gomhoreya Co., Egypt. Donepezil hydrochloride, is the main active
ingredient in Donazil®; it acts as a selective acetylcholinesterase
inhibitor and hence, enhancing the cholinergic activity in the brain
which is insufficient with AD (Seltzer, 2005). Donazil was purchased
from Eva Pharma, Egypt, and was dissolved in Carboxy Methyl
Cellulose (CMC) (0.25%) and administered orally.
Animals
Forty-two male albino rats, weighting 200 ± 20 g at the start of the
experiments were used. Prior to the initiation of the studies, the
animals were randomized and assigned to treatment groups. Four
rats were housed per cage (size 26 × 41 cm) and placed in the
experimental room for acclimatization 24 h before the test. The
animals were fed with standard laboratory diet and with tap water
ad libitum, and kept in an air-conditioned animal room at 23 ±1°C
with a 12 h light/dark cycle. Animal care and handling was
performed in conformity with approved protocols of MSA University,
Faculty of Pharmacy, Research Ethics Committee and Egyptian
Community guidelines for animal care.
Experimental groups
The dose of BM extract was determined according to methodology
of Sathiyanarayanan et al. (2010). The LD50 of RO extract was
determined previously (Anadon et al., 2008) which is 2000 mg/kg of
body weight. Rats were randomly allocated into seven groups of six
animals each. Rats were orally given the hydro-ethanolic extracts of
BM and RO for a period of 2 weeks prior to injection of AlCl3.
Group 1: Control group injected (I.P) by 0.9% saline.
Group 2 (AlCl3): Rats injected AlCl3 at dose 4.2 mg/kg/day (I.P) for
28 days.
Group 3 (BM 300): Rats received BM extract 300 mg/kg/day, orally
for 28 days and injected by the same dose of AlCl3 for 28 days.
Group 4 (BM 450): Rats received BM extract 450 mg/kg/day, orally
for 28 days and injected by the same dose of AlCl3 for 28 days.
Group 5 (RO 300): Rats received RO extract 300 mg/kg/day, orally
for 28 days and injected by the same dose of AlCl3 for 28 days.
Group 6 (RO 450): Rats received RO extract 450 mg/kg/day, orally
for 28 days and injected by the same dose of AlCl3 for 28 days.
Group 7 (Donazil® 500): Rats received Donazil 2.5 mg/kg/day,
orally for 28 days and injected by the same dose of AlCl3 for 28
days.
Blood samples and biochemical analysis
Preparation of blood samples
At the end of the study, rats were fasted overnight, anesthetized
with thiopental sodium (50 mg/kg) (Vogler, 2006) and blood
samples were collected in the morning (5 ml per rat). Blood
samples were centrifuged at 3000 rpm for 15 min after 30 min of
collection and stored at –80°C until analyzed for the analysis of
Malondialdehyde (MDA), and Total anti-oxidant capacity (TAC).
Preparation of brain samples
Animals were euthanized by cervical dislocation, and then the brain
was rapidly removed from each rat. Part of each brain was fixed in
formalin-saline for 48 h for histopathological study. Another part of
the brain was homogenized, using glass homogenizer (Universal
Lab. Aid MPW-309, mechanika precyzyjna, Poland), with 5 ml
phosphate buffer saline (PBS) then centrifuged using cooling ultra-
centrifuge. The homogenate was divided into four aliquots for
measuring Amyloid β (Aβ) - peptide, acetylcholinesterase (AChE),
tau (τ) protein and serotonin.
Biochemical analysis
Analysis of serum was carried out for measuring MDA, and TAC
levels using corresponding colorimetric Cell Biolabs, Inc, USA and
Zen-Bio, ABTS antioxidant assay kit, Inc., USA respectively. Aβ-
peptide, tissue AChE, τ-protein and serotonin were measured using
corresponding rat enzyme immunoassay kits Wuhan EIAAB
Science Co, Ltd, China, Kamiya Biomedical Company, USA,
Elabscience, USA, and Lifespan Biosciences Inc. USA, respectively.
Histopathological examination
As mentioned earlier, brain tissue was fixed in 10% formalin and
then routinely processed and embedded in paraffin. Five microns’
sections were cut and stained with hematoxylin and Eosin (H&E)
and Congo Red.
Statistical analyses
All data were expressed as mean ± SD and analyzed using Prism
program version 6. For all parameters, comparisons among groups
were carried out using one-way analysis of variance (ANOVA)
followed by Bonferroni’s multiple comparisons test. All P values
reported are two-tailed and P < 0.05 was considered significance.
Ethics approval
Animal care and handling was performed in conformity with
approved protocols of MSA University and Egyptian Community
guidelines for animal care.
RESULTS AND DISCUSSION
Characterization of major metabolites in BM and RO
extracts via UPLC-ESI-MS
Chromatographic fingerprints of the hydro-ethanolic
extracts of BM and RO were obtained using UPLC-ESI-
MS as depicted in Figures 1 and 2 respectively. The
identities, elemental compositions, relative percentages
and observed molecular and product ions for individual
components in both positive and negative modes are
presented in Tables 1 and 2 respectively. With the
optimized LC and MS conditions, a total of 15 and 17
Waly et al. 255
Figure 1. The LC–ESI/MS Chromatograms of the hydro-ethanolic extracts of B. monnieri (A): positive mode and
(B): negative mode.
metabolites were tentatively characterized in BM and RO
extracts respectively on the basis of their elemental
compositions and MS fragmentation patterns compared
to the data previously reported in literature.
The major metabolites of BM are dammarane-type of
steroidal saponins that are categorized into jujubogenin
saponin glycosides and pseudojujubogenin saponin
glycosides. Pseudojujubogenin is the isomer of
jujubogenin having different position of the prenyl side
chain (Nuengchamnong et al., 2016), in addition to,
cucurbitacins and sterol glycosides. The UPLC-ESI-MS
analyses revealed the presence of 5 jujubogenin saponin
glycosides, namely; bacoside A1, bacopasaponin F,
bacopaside N1, deoxy-jujubogenin-Ara-Glc and deoxy-
jujubogenin-2-Glc; and 7 pseudojujubogenin saponin
glycosides, namely; bacopaside (I, II and III), oxy-
bacopaside I, bacopasaponin (C and D) and
pseudojujubogenin-Glc-Glu-Ara. Cucurbitacins
(bacobitacin B and C) and bacosterol-Glc were also
identified in the hydro-ethanolic extract of BM.
The UPLC-ESI-MS analyses of RO hydro-ethanolic
extracts led to the characterization of rosemary
landmarks represented in 6 phenolic diterpenes, namely;
carnosol, carnosic acid, rosmanol, epirosmanol,
rosmadial and methyl carnosate, 3 phenolic acids
namely; Gallic acid, caffeic acid and rosmarinic acid.
Flavonoids of flavone and flavanone types were also
detected such as apigenin, luteolin, cirsimaritin,
hesperidin and their glycosides, in addition to the
dihydrochalcone (phloridzin) and the lignan
(medioresinol).
Effect of Bacopa monnieri and Rosmarinus officinalis
extracts on serum MDA and TAC levels
Mean serum level of MDA was significantly increased in
AlCl3 induced Alzheimer’s group compared to the control
group (P value was < 0.001). Mean serum level of MDA
was significantly reduced in BM 300, BM 450, RO 300,
256 J. Med. Plants Res.
Figure 2. The LC–ESI/MS Chromatograms of the hydro-ethanolic extracts of R. officinalis (A): positive mode and (B): negative
mode.
RO 450, and Donazil groups compared to AlCl3 induced
Alzheimer’s group (P value < 0.001). RO 450 treatment
reduced MDA level compared to BM 450 and Donazil
groups (P value was < 0.001).
Similarly, mean serum level of TAC was significantly
decreased in AlCl3 induced Alzheimer’s group compared
to the control group (P value was < 0.0001). The mean
serum level of TAC was significantly raised in BM 300,
BM 450, RO 300, RO 450, and Donazil groups compared
to AlCl3 induced Alzheimer’s group (P value < 0.001).
TAC was increased in RO 450 group compared to
Donazil and BM 450 groups at P value < 0.01 (Figures 3
and 4; Table 3).
Effect of Bacopa monnieri and Rosmarinus officinalis
extracts on tissue amyloid beta protein and τ-protein
levels
Mean tissue level of amyloid β peptide was significantly
increased in AlCl3 induced Alzheimer’s group compared
to control group (P value was < 0.0001). On the other
hand, mean tissue level of amyloid β peptide was
significantly reduced in BM 300, BM 450, RO 300, RO
450, and Donazil groups compared to AlCl3 induced
Alzheimer’s group (P value < 0.0001). Amyloid β peptide
was decreased in RO 450 group compared to Donazil and
BM 450 groups at P value < 0.01 (Figure 5 and Table 4).
Waly et al. 257
Table 1. Peak assignment of metabolites in the hydro-ethanolic extract of Bacopa monnieri using LC–ESI/MS in the positive and negative modes.
Peak No.
Positive Ionization
Negative Ionization
Elemental
composition
Tentative compound
assignment
Relative (%)
References
[M+H]+ (m/z)
Product ion
fragments (m/z)
[M-H]- (m/z)
Product ion
fragments (m/z)
Jujubogenin saponin glycosides
1
736.69
455
n.d.
n.d.
C40H64O12
Bacoside A1
2.65
Nuengchamnong et al.
(2016)
2
n.d.
n.d.
1059.38
765, 633
C52H84O22
Bacopasaponin F
2.13
3
797.47
779 , 635, 455
795.39
633
C42H68O14
Bacopaside N1
1.47
4
749.43
437
747.32
435
C41H64O12
Deoxy-Jujubogenin-Ara-Glc
4.39
5
779.62
437
n.d.
n.d.
C46H66O10
Deoxy- Jujubogenin -2 Glc
2.81
Pseudojujubogenin saponin glycosides
6
979.1
767, 605, 473
977.46
845, 241
C46H74O20S
Bacopaside I
32
Sookying et al. ( 2017)
7
n.d.
n.d.
927.48
795, 633
C47H76O18
Bacopaside II
5.11
Nuengchamnong et al.
(2016)
8
847.41
765, 515, 393
n.d.
n.d.
C41H66O16S
Bacopaside III
14.18
9
n.d.
n.d.
993.51
861, 505, 389
C46H74O21S
Oxy-Bacopaside I
4
10
n.d.
n.d.
897.03
765, 603
C46H74O17
Bacopasaponin C
3.5
11
767.5
605, 473
n.d.
n.d.
C41H66O13
Bacopasaponin D
7.71
12
n.d.
n.d.
941.53
809, 647
C47H74O19
Pseudojujubogenin-Glc-Glu-Ara
28.06
Cucurbitacins
13
599.37
479, 443
n.d.
n.d.
C34H46O9
Bacobitacin B
6.23
Bhandari et al. (2006)
14
1113.47
981, 835
n.d.
n.d.
C54H80O24
Bacobitacin C
17.5
Sterol glycosides
15
575.89
414
n.d.
n.d.
C35H60O6
Bacosterol-Glc prevent
9.72
Bhandari et al. (2006)
*n.d.: Not detected, Glc: glucose, Glu: glucouronide, Ara: arabinose.
Similarly, mean tissue level of tau-protein was
significantly increased in AlCl3 induced
Alzheimer’s group compared to control group (P
value < 0.001). Also, the mean tissue levels of τ -
protein was significantly reduced in BM 300, BM
450, RO 300, RO 450, and Donazil groups
compared to AlCl3 induced Alzheimer’s group (P
value < 0.001). τ -protein was decreased in RO
450 group compared to Donazil and BM 450
groups at P value < 0.01 (Figure 8 and Table 5).
Effect of Bacopa monnieri and Rosmarinus
officinalis extracts on tissue
acetylcholinesterase (AChE) levels
Mean tissue levels of AChE were significantly
increased in AlCl3 induced Alzheimer’s group
compared to control group (P value < 0.0001).
AChE were significantly reduced in BM 300, BM
450, RO 300, RO 450, and Donazil groups
compared to AlCl3 induced Alzheimer’s group (P
value < 0.0001). Also, AChE was reduced in RO
450 group compared to Donazil and BM 450
groups at P value < 0.0001 (Figure 6).
258 J. Med. Plants Res.
Table 2. Peak assignment of metabolites in the hydro-ethanolic extract of Rosmarinus officinalis using LC–ESI/MS in the positive and negative modes.
Peak No.
Positive Ionization
Negative Ionization
Elemental
composition
Tentative compound
assignment
Relative
(%)
References
[M+H]+ (m/z)
Product ion
fragments (m/z)
[M-H]- (m/z)
Product ion
fragments (m/z)
Phenolic diterpenes
1
331.34
287
n.d.
n.d.
C20H25O4
Carnosol
8.32
Hossain et al. (2010)
2
n.d.
n.d.
331.24
287, 244
C20H27O4
Carnosic acid
19.47
3
347.19
285
345.25
283
C20H26O5
Rosmanol
1.87
Achour et al. (2018)
4
347.18
285
345.16
283
C20H26O5
Epirosmanol
0.85
Borras-Linares et al. (2014)
5
n.d.
n.d.
343.37
315, 300
C20H23O5
Rosmadial
16.28
Hossain et al. (2010)
6
347.2
303, 288
n.d.
n.d.
C21H29O4
Methyl carnosate
0.72
Phenolic acids
7
171.01
127
169.03
125
C7H5O5
Gallic acid
0.93
Hossain et al. (2010)
8
n.d.
n.d.
179.12
161, 135
C9H7O4
Caffeic acid
0.95
9
n.d.
n.d.
359.07
197, 161
C18H16O8
Rosmarinic acid
19.39
Borras-Linares et al. (2014)
Flavonoids: Flavones
10
n.d.
n.d.
269.1
269
C15H10O5
Apigenin
3.75
Achour et al. (2018)
11
n.d.
n.d.
576.95
269
C27H29O14
Apigenin-7-O-rutinoside
5.36
Hossain et al. (2010)
12
n.d.
n.d.
285.05
263, 191
C15H10O6
Luteolin
24.36
Achour et al. (2018)
13
n.d.
n.d.
478.91
315
C22H22O12
Nepetrin
1.5
Borras-Linares et al. (2014)
14
n.d.
n.d.
313.09
298, 283
C17H13O6
Cirsimaritin
12.19
Flavonoids: Flavanone
15
611.07
463, 303
n.d.
n.d.
C28H34O15
Hesperidin
20.24
Achour et al (2018)
Dihydrochalcone
16
437.15
275, 169
435.13
273, 167
C21H23O10
Phloridzin
0.73
Hossain et al. (2010)
Lignan
17
389.23
209, 165
n.d.
n.d.
C21H24O7
Medioresinol
1.28
Mena et al. (2016)
*n.d.: Not detected.
Effect of Bacopa monnieri and Rosmarinus
officinalis extracts on tissue serotonin level
Mean tissue level of serotonin was significantly
decreased in AlCl3 induced Alzheimer’s group
compared to control group (P value < 0.0001).
Also, mean tissue levels of serotonin was
significantly elevated in BM 300, BM 450, RO 300,
RO 450, and Donazil groups compared to AlCl3
induced Alzheimer’s group (P value < 0.0001).
Serotonin was increased in RO 450 group
compared to Donazil and BM 450 groups at P
Waly et al. 259
Table 3. Effect of Bacopa monnieri and Rosmarinus officinalis extracts on serum MDA and TAC levels.
Groups
Serum MDA (umol/ml)
Serum TAC ( umol/ml )
Control
12.4 ± 1.40
100 ± 7.43
ALZ
38 ± 4.56 a
41.9 ± 3.80 a
BM 300
26.5 ± 0.87 ab
53 ± 3.80 ab
BM 450
20.9 ± 1.24 abc
68 ± 3.82 abc
RO 300
32.5 ± 3.66 ab
54.4 ± 4.39 ab
RO 450
17.7 ± 1.32 ab
84.2 ± 5.52 ab
Donazil
21.4 ± 1.28 abc
76.7 ± 4.94 abc
C = control; ALZ = Alzheimer’s; BM 300 = Bacopa monnieri extract 300 mg/kg; BM 450 = Bacopa monnieri 450
mg/kg; RO 300 = Rosmarinus officinalis extract 300 mg/kg; RO 450 = Rosmarinus officinalis extract 450 mg/kg;
D = Donazil. 2.5 mg/kg. Results were expressed as mean ± SD and analyzed using one-way ANOVA followed
by Bonferroni’s post hoc test a = Significant from control at P < 0.001, b = Significant from ALZ at P < 0.001, c =
Significant from RO 450 at P < 0.001.
Table 4. Effect of Bacopa monnieri and Rosmarinus officinalis extract on tissue amyloid beta protein and
acetylcholinestrase levels.
Groups
Tissue amyloid beta peptide
(Pg/gm tissue)
Tissue acetylcholinestrase
(ng/gm tissue)
Control
9.3 ± 0.92
0.81 ± 0.08
ALZ
30.9 ± 4.01a
3.14 ± 0.26a
BM 300
21.8 ± 1.37ab
2.2 ± 0.08 ab
BM 450
17.3 ± 1.09abc
1.79 ± 0.08 abc
RO 300
21.8 ± 1.74ab
2.34 ± 0.15 ab
RO 450
13.2 ± 0.95ab
1.21 ± 0.11 ab
Donazil
18.9 ± 0.9abc
1.67 ± 0.11 abc
C = control; ALZ = Alzheimer’s; BM 300 = Bacopa monnieri extract 300 mg/kg; BM 450 = Bacopa monnieri 450 mg/kg;
RO 300 = Rosmarinus officinalis extract 300 mg/kg; RO 450 = Rosmarinus officinalis extract 450 mg/kg; D = Donazil.
2.5 mg/kg. Results were expressed as mean ± SD and analyzed using one-way ANOVA followed by Bonferroni’s post
hoc test a = Significant from control at P < 0.0001, b = Significant from ALZ at P < 0.0001, c = Significant from RO 450
at P < 0.01.
value < 0.0001 (Figure 7).
Histopathological changes associated with herbal
treatment
Brain sections from the control group (C) showed normal
histological appearance in both the neurons and the
blood vessels. The ALZ group showed marked neuronal
degenerative changes and marked amyloid deposits on
the blood vessels. All other groups showed different
histological changes in both neurons and blood vessels
illustrated in Figures 9 and 10.
The present study showed that Bacopa monnieri (BM)
and Rosmarinus officinalis (RO) have a neuroprotective
effect in AD rat model. This effect is possibly mediated
via anti-AChE and antioxidant, and monoaminergic
pathways modulation. Both extracts are potential
candidates in the management of AD.
BM significantly increased serum TAC levels on the
other hand significantly reduced MDA, a well-known
oxidative stress biomarker, in AlCl3 induced AD rat
model. This clearly indicates the enhancement of the
antioxidant activity, which is a key component in AD
pathogenesis (Bartus, 2000). This result comes in
agreement with several studies that have shown that BM
exerts antioxidant activity both in vivo and in vitro (Russo
et al., 2003; Bhattacharya et al., 2000; Chaudhari et al.,
2017). Jyoti et al. (2007) reported that BM inhibited the
reduction of superoxide dismutase (SOD) activity in AlCl3
induced neurotoxicity of rat brain. It also showed that BM
inhibited thiobarbituric acid reactive substance (TBARS),
an index of lipid peroxidation (LPO) and its accumulation
associated with AlCl3 neurotoxicity of rat brain (Jyoti et
al., 2007; Jyoti and Sharma, 2006). Our results further
elaborate and confirm these reports of the antioxidant
effect of BM as a neuroprotective agent in AD that is
comparable to current medications used (Figures 3 and
4). In addition, it was shown that BM reduced Aβ and τ -
protein levels in AD rat model. It was also observed that
260 J. Med. Plants Res.
Table 5. Effect of Bacopa monnieri and Rosmarinus officinalis extract on tissue serotonin in Alzheimer’s rats.
Groups
Tissue serotonin
(ng/gm tissue)
Tissue Tau-protein
(Pg/gm tissue)
Control
47.7 ± 6.27
8.2 ± 1.02
ALZ
6.07 ± 0.77 a
30.7 ± 2.91 a
BM 300
23.6 ± 2.31 ab
23.5 ± 0.97 ab
BM 450
21.9 ± 3.24 abc
17.3 ± 1.17 abc
RO 300
12.8 ± 1.22 ab
22.8 ± 1.78 ab
RO 450
37.5 ± 2.93 ab
14.7 ± 1.14 ab
Donazil
14.9 ± 0.73 abc
17.3 ± 1.42 abc
C = control; ALZ = Alzheimer’s; BM 300 = Bacopa monnieri extract 300 mg/kg; BM 450 = Bacopa monnieri 450 mg/kg;
RO 300 = Rosmarinus officinalis extract 300 mg/kg; RO 450 = Rosmarinus officinalis extract 450 mg/kg; D = Donazil.
2.5 mg/kg. Results were expressed as mean ± SD and analyzed using one-way ANOVA followed by Bonferroni’s post
hoc test a = Significant from control at P < 0.0001, b = Significant from ALZ at P < 0.0001, c = Significant from RO 450
at P < 0.0001.
Figure 3. Serum level of MDA (µmol/ml) in the experimental groups. BM and RO extract reduced serum level of MDA in
Alzheimer’s rats at the end of 2-month prophylaxis; C = control; ALZ = Alzheimer’s; BM 300 = B. monnieri extract 300 mg/kg;
BM 450 = B. monnieri 450 mg/kg; RO 300 = R. officinalis extract 300 mg/kg; RO 450 = R. officinalis extract 450 mg/kg; D =
Donazil. 2.5 mg/kg. Results were expressed as mean ± SD and analyzed using one-way ANOVA followed by Bonferroni’s
post hoc test a = Significant from control at P < 0.001, b = Significant from ALZ at P < 0.001, c = Significant from RO 450 at P
< 0.001.
BM at tested doses reduced Aβ and τ -protein levels in
AD rat model brain when examined histologically using
H&E and Congo Red staining compared to control.
Neurons exhibited unremarkable degenerative changes
while blood vessels showed moderate and unremarkable
amyloid thickening (Figures 9 and 10).
We also demonstrated that BM extract inhibited
significantly acetylcholinesterase (AChE) activity in AD
rat model, a result that further support its use as a
neuroprotective agent in AD. It is well documented that
cholinergic dysfunction is implicated in AD pathogenesis
although the mechanism is not well understood (Bartus,
2000). Our results support the hypothesis that the
observed neuroprotective effect of BM extract can be
attributed to inhibition of AChE consequently preserving
ACh longer at the synapses and compensating for the
E ffe ct o f B ac op a m o nn ieri and R osm ar in us officin alis ex tr act o n seru m M D A
of A L Z m o de l in r ats
C o ntro l
AL Z
BM 3 00
BM 4 50
RO 3 00
RO 4 50
Donazil
0
10
20
30
40
50 a
a b
a b c
a b
a b a b c
M D A (u m ol/m l)
Waly et al. 261
Figure 4. Serum level of TAC (µmol/ml) in the experimental groups. BM and RO extract raised serum level of TAC in
Alzheimer’s rats at the end of 2 month prophylaxis; C = control; ALZ = Alzheimer’s; BM 300 = B. monnieri extract 300
mg/kg; BM 450 = B. monnieri 450 mg/kg; RO 300 = R. officinalis extract 300 mg/kg; RO 450 = R. officinalis extract 450
mg/kg; D = Donazil. 2.5 mg/kg. Results were expressed as mean ± SD and analyzed using one-way ANOVA followed by
Bonferroni’s post hoc test a = Significant from control at P < 0.0001, b = Significant from ALZ at P < 0.001, c = Significant
from RO 450 at P < 0.01.
Figure 5. Tissue level of amyloid beta peptide (Pg/gm tissue) in the experimental groups. BM and RO extract reduced
tissue level of amyloid beta in Alzheimer’s rats at the end of 2-month prophylaxis; C = control; ALZ = Alzheimer’s; BM
300 = B. monnieri extract 300 mg/kg; BM 450 = B. monnieri 450 mg/kg; RO 300 = R. officinalis extract 300 mg/kg; RO
450 = R. officinalis extract 450 mg/kg; D = Donazil. 2.5 mg/kg. Results were expressed as mean ± SD and analyzed
using one-way ANOVA followed by Bonferroni’s post hoc test a = Significant from control at P < 0.0001, b = Significant
from ALZ at P < 0.0001, c = Significant from RO 450 at P < 0.01.
E ffe ct of B acopa m on nie ri and R osm arin us o fficinalis extr act on serum
T A C o f A LZ m od el in rats
C o ntr ol
A L Z
B M 300
BM 4 50
RO 3 00
RO 4 50
Donazil
0
50
100
150
aa b a b c a b
a b a b c
T A C u m o l/m l
E ffe ct of B acopa m on nieri an d R osm a rinus o ffic in alis extra ct on tiss ue
am ylo id b eta p ep tid e o f A LZ m o de l in rats
C o ntr ol
A L Z
BM 300
BM 4 50
RO 3 00
RO 4 50
Donazil
0
10
20
30
40 a
a b
a b c a b c
a b
a b
A m ylo id b e ta P g /g m t is su e
262 J. Med. Plants Res.
Figure 6. Tissue level of acetyl cholinestrase (ng/gm tissue) in the experimental groups. BM and RO extract
reduced tissue level of amyloid beta in Alzheimer’s rats at the end of 2-month prophylaxis; C = control; ALZ =
Alzheimer’s; BM 300 = B. monnieri extract 300 mg/kg; BM 450 = B. monnieri 450 mg/kg; RO 300 = R.
officinalis extract 300 mg/kg; RO 450 = R. officinalis extract 450 mg/kg; D = Donazil. 2.5 mg/kg. Results were
expressed as mean ± SD and analyzed using one-way ANOVA followed by Bonferroni’s post hoc test a =
Significant from control at P < 0.0001, b = Significant from ALZ at P < 0.0001, c = Significant from RO 450 at P
< 0.0001.
Figure 7. Tissue level of serotonin (ng/gm tissue) in the experimental groups. BM and RO extract raised tissue
level of serotonin in Alzheimer’s rats at the end of 2 -month prophylaxis; C = control; ALZ = Alzheimer’s; BM 300 =
B. monnieri extract 300 mg/kg; BM 450 = B. monnieri 450 mg/kg; RO 300 = R. officinalis extract 300 mg/kg; RO
450 = R. officinalis extract 450 mg/kg; D = Donazil. 2.5 mg/kg. Results were expressed as mean ± SD and
analyzed using one-way ANOVA followed by Bonferroni’s post hoc test a = Significant from control at P < 0.0001,
b = Significant from ALZ at P < 0.0001, c = Significant from RO 450 at P < 0.0001.
E ffe ct of B ac op a m o nn ieri an d R osm a rinus o fficin alis extr act on tissu e
acetylch olinestra se of A L Z m o del in rats
C o ntro l
AL Z
B M 300
BM 4 50
R O 3 00
RO 4 50
Donazil
0
1
2
3
4a
a b a b c
a b
a b
a b c
A c e ty lc h oli ne st ra se n g /g m ti ss u e
E ffe ct o f B acopa m o nnieri a nd R osm ar in us officinalis extr act on tiss ue
se roto nin of A L Z m o de l in rats
C o ntrol
AL Z
BM 3 00
B M 450
RO 3 00
RO 4 50
Donazil
0
20
40
60
S er o to n in (n g /g m t is su e )
a b a b c
a
a b
a b
a b c
Waly et al. 263
E ffect of B ac op a m o nn ieri a nd R osm ar in us o ffic in alis e xtra ct on tis su e
T au -p ro te in o f A L Z m o de l in rats
T a u -P r o te in ( P g /g m ti ss u e
C o ntro l
A L Z
BM 3 00
BM 4 50
RO 3 0 0
R O 4 50
Donazil
0
10
20
30
40 a
ab
a b c
ab
ab a b c
Figure 8. Tissue level of Tau-Protein (Pg/gm tissue) in the experimental groups. BM and RO extract reduced
tissue level of tau-protein in Alzheimer’s rats at the end of 2-month prophylaxis; C = control; ALZ =
Alzheimer’s; BM 300 = B. monnieri extract 300 mg/kg; BM 450 = B. monnieri 450 mg/kg; RO 300 = R.
officinalis extract 300 mg/kg; RO 450 = R. officinalis extract 450 mg/kg; D = Donazil. 2.5 mg/kg. Results were
expressed as mean ± SD and analyzed using one-way ANOVA followed by Bonferroni’s post hoc test a =
Significant from control at P < 0.001, b = Significant from ALZ at P < 0.001, c = Significant from RO 450 at P <
0.01.
lost cholinergic function. To our knowledge, this is the
first demonstration of BM effect on AChE.
The mechanism underlying the protection of Brahmi
against Aβ25–35-mediated neurotoxicity demonstrated
that Aβ 25–35 induced neurotoxicity causes the elevation
of intracellular AChE activity and so the elevation of
AChE activity was diminished by co-treatment of cortical
cells with Brahmi extract. Also, AChE was proved to be
neurotoxic both in vitro and in vivo models. This
observation suggests the neuroprotection of Brahmi
through its inhibitory effect on amyloid peptide-activated
intracellular AChE activity (Limpeanchoba et al., 2008).
The presence of the nootropic metabolite; Bacopaside I
(Pseudojujubogenin- 3-O-[α-l-arabinofuranosyl-(1→2)]-6-
O- sulfonyl-α-D-glucopyranosyl-(1→3)-α-l-
arabinopyranoside) is believed to account for the
neuroprotective effect of BM extract as it reverses the
depressive-like symptoms caused by reserpine, which is
mediated through antioxidant and noradrenergic
activations. It also stimulates PI3/Akt signaling in
organotypic hippocampal slice cultures (Yin et al., 2016).
In accordance, a study conducted by Le et al. (2015)
concluded that bacopaside I played a role in
neuroprotective effects in both in vitro and in vivo, where
in the in vitro experiment, the hippocampal slice cultures
(OHSCs) were incubated with triterpenoid saponins from
BM, where bacopaside I exhibited potent neuroprotective
effects against OGD-induced neuronal cell damage (Le et
al., 2015). However, the role of each dammarane
steroidal saponin of BM in the neuroprotection is still
uncovered.
The presence of Bacoside metabolite is thought to
have anti-oxidant and free radical scavenging as it
inhibits lipid peroxidation and elevates the anti-oxidant
enzymes in prefrontal cortex, hippocampus, and striatum
which also possess a significant iron chelating property;
also, iron and other divalent metals interact with Aβ
protein and modulate several effects that are thought to
be the pathogenic effects of that protein (Chaudhari et al.,
2017).
Similar to BM, R. officinalis (RO) extract also exhibited
antioxidant effects in our AlCl3 induced AD rat model.
This effect was demonstrated by its significant reduction
of the oxidative stress biomarker MDA and enhancement
of TAC (Figures 3 and 4). This result complement
previous reports that Carnosic acid (CA), a metabolite
found in rosemary and sage, had antioxidant effects in
both in vitro and transgenic mice (Lipton et al., 2016).
Also, Rasoolijazi et al. (2013) reported on its role in
memory and learning scores improvement. CA decreased
264 J. Med. Plants Res.
Figure 9. Histopathological sections of brain tissue illustrating herbal treatment effect in
all experimental groups. A: negative control, illustrates unremarkable changes in both
neurons and vessels (V). B: ALZ group exhibiting neuronal marked degenerative
changes (thin arrow). C: RO 300 group showed minimal neuronal degenerative changes
(thin arrow). D: RO 450 group showed unremarkable neuronal degenerative changes
(thin arrow). E: BM 300 group demonstrating un remarkable neuronal degenerative
changes (thin arrow). F: BM 450 group demonstrating un remarkable neuronal
degenerative changes (thin arrow). G: Donazil group showing unremarkable
degenerative changes of neurons (thin arrows). H&E staining. Magnification 200, arrow
pointing at neurons, V blood vessels.
52% of the infarct volume from brains under
ischemia/reperfusion in vivo, and also protected the PC12 cells from hypoxic injury via reducing oxidative
stress biomarkers and enhancing cell viability in vitro
Waly et al. 265
Figure 10. Histopathological sections of brain tissue illustrating herbal treatment
effect in all experimental groups. A: negative control illustrates unremarkable
changes in both neurons and vessels (V). B: ALZ group exhibited marked
thickening of the vessel wall by the amyloid deposits on blood vessels (thick
arrow). C: RO 300 group showed mild thickening of the vessel wall by the
amyloid deposits (thick arrow). D: RO 450 group showed moderate thickening
of the vessel wall by the amyloid deposits (thick arrow). E: BM 300 group
exhibiting moderate thickening of the vessel wall by the amyloid deposits (thick
arrow). F: BM 450 group exhibiting unremarkable thickening of the vessel wall
by the amyloid deposits (thick arrow). G: Donazil group showing unremarkable
thickening of the vessel wall by the amyloid deposits (thick arrow). Congo Red
staining. Magnification 200, arrow pointing at neurons, V blood vessels.
A
B
C
D
E
F
G
266 J. Med. Plants Res.
(Hou et al., 2012).
Not only that CA has a nootropic effect, but also, the
administration of Rosmarinic acid (RA) averted cognitive
impairment induced by chronic ethanol (Hasanein et al.,
2017). Actually, the mechanism of action of RO
pertaining to neuroprotection could also be attributed to
the synergistic effects of phenolic and polyphenolic
metabolites that possess well-known antioxidant and
anticholinesterase activities.
Our experiments also showed that RO at selected
doses reduced Aβ and τ -protein levels in AD rat model
brain, a result that comes in agreement with a previous
report of the effect of CA on U373MG human
astrocytoma cells probably via activation of α-secretase
(Yoshida et al., 2014). This may also explain our
histopathological examination of RO treated rat brains
that revealed mild to unremarkable degenerative changes
as well as mild to moderate amyloid thickening of
vascular walls (Figures 9 and 10).
Since there seems to be an agreement that AD
pathogenesis results primarily from defective brain
cholinergic function (Bartus, 2000; Iqbal and Grundke-
Iqbal, 2010; Murphy and Steenbergen, 2008), we
investigated the possibility that RO neuroprotective
effects in our AD rat model may be due to enhancement
of cholinergic function. It was found that, similar to BM,
RO significantly inhibited AChE. This also agrees with
previous report that RO improved long-term memory and
inhibited the AChE activity of rat brain (Ozarowski et al.,
2013). These results may also explain the observed
effectiveness of rosemary aromatherapy in human (Jimbo
et al., 2009).
It is well-known that nitrocatechol derivatives exhibit
anti-aggregation properties against Aβ protein. It is also
documented that Rosmarinic acid had two cathecol
moieties, which consequently induces morphological and
signature changes in the secondary structure of tau-
protein once it is interacted with, thus, preventing
aggregation and β-sheets assembly and also reducing
fibril progression (Cornejo et al., 2015). Both RO and BM
effects on all afore-mentioned biochemical markers and
histopathological results were comparable to Donazil®™,
a standard selective inhibitor of brain cholinesterase
commonly used in AD management (Bitra et al., 2014;
Nayak and Chatterjee, 2001).
Serotonin (5-hydroxytryptamien, 5-HT) has been linked
to emotional and motivational aspects of human behavior
and memory (Meneses and Liy-Salmeron, 2012).
Recently, it has been documented that the serotonin as a
neurotransmitter is involved in the pathophysiology of AD
(Ramirez, 2013; Maccioni et al., 2018). Hence, the 5-
HT6 receptor is a promising target for cognitive disorders
AD (Amat-Foraster et al., 2017) where we aimed to
evaluate tissue serotonin in both our AD model and with
herbal therapy (Figure 7).
Indeed, our results have shown the diminution of tissue
serotonin as well as its partial restoration with our herbal
treatment.
Both extracts exhibited significant elevation of tissue
serotonin levels with a prominent effect for RO 450 mg/kg
compared to the controls. This result is the first
demonstration that RO and BM extracts may exert their
neuroprotective effects in AD rat model via serotoninergic
system. It was previously reported that RO extract
exhibited antidepressant-like activity in mice via the
monoaminergic system (Machado et al., 2009). BM, on
the other hand, elevated serotonin levels rats subjected
to stress (Sheikh et al., 2007). Our results is in line with
other reports of BM and RO influencing the
monoaminergic system (Rajan et al., 2015; Machado et
al., 2009), however, we are the first to report this effect in
AD rat model.
Since both BM and RO have similar antioxidant and
monoaminergic effects, combining both RO and BM can
have synergistic effects in the treatment of AD.
Ramachandra et al. (2014) reported its neuroprotective
effectiveness in contrast to its individual use in embryonic
cell line (Ramachandran et al., 2014). The effect in
animal model still needs to be investigated.
This study showed that B. monnieri (BM) and R.
officinalis (RO) have a neuroprotective effect in AD rat
model at both histological and biochemical levels which is
due to their interesting variety of bioactive metabolites.
This effect is possibly mediated via anti-AChE,
monoaminergic and antioxidant pathways modulation.
Further pharmacological and clinical studies are needed
to confirm these results.
CONFLICT OF INTERESTS
The authors have not declared any conflict of interests.
ACKNOWLEDGEMENTS
The authors appreciate Dr. Hebatallah Amin, Lecturer of
Pathology, Helwan University for the invaluable technical
assistance of preparing the histopathological sections
micrographs, as well as Dr. Nashwa Waly, Professor of
Small Animal Medicine, Assiut University, Egypt, for the
invaluable editorial assistance.
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