Investigation of therapeutic effect of curcumin α and β glucoside anomers against Alzheimer's disease by the nose to brain drug delivery

Article abstract of DOI:10.1016/j.brainres.2021.147517

Alzheimer's disease (AD) is one of the greatest geriatric medicinal challenges of our century and is the main disease leading to dementia. Despite extensive scientific research advances, available disease-modifying treatment strategies remained limited; t

Full text of DOI:10.1016/j.brainres.2021.147517

Brain Research 1766 (2021) 147517
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Brain Research
journal homepage: www.elsevier.com/locate/brainres
Investigation of therapeutic effect of curcumin
α
and β glucoside anomers
against Alzheimer’s disease by the nose to brain drug delivery
Nahid Ahmadia, Mir-Jamal Hosseinib, Kobra Rostamizadehc, Mahdieh Anoushb
,d,*
a Department of Medicinal Chemistry, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran
b Zanjan Applied Pharmacology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran
c Zanjan Pharmaceutical Nanotechnology Research Center, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran
d Department of Pharmacology and Toxicology, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran
A R T I C L E I N F O
A B S T R A C T
Keywords:
Alzheimer’s disease (AD) is one of the greatest geriatric medicinal challenges of our century and is the main
disease leading to dementia. Despite extensive scientific research advances, available disease-modifying treat-
ment strategies remained limited; thus, increasing demand for new drugs. In recent years, medicinal plants
Curcumin
Glucoside
Glucosylation
Fusion reactions
Intranasal
attracted attention due to their potential role in dementia. In the present study,
α and β anomers of curcumin
glucosides (CGs) were synthesized and evaluated for Alzheimer’s treatment. CGs were synthesized by fusion
reaction as a novel and easy method with more advantages (high yield, short reaction time, and low chemicals),
and the products were characterized using HNMR. Wistar male rats were used to administer different treatments.
Alzheimer’s disease
They divided into control, sham, Alzheimer, and test groups (Alzheimer +
α anomer and Alzheimer + β anomer).
Animals received normal saline, Scopolamine (1 mg/kg), high dose anomers, scopolamine, and two doses (12.5
and 25 mg/kg) of anomers, respectively, for 10 days. Then the Morris Water Maze (MWM) test was performed on
all animals. Finally, the animals’ brains were extracted and homogenized for glutathione, acetylcholine esterase
activity, protein carbonyl, and lipid peroxide level detection. The escape latency and the distance towards the
hidden platform in Morris water maze in the Alzheimer group were significantly higher than both the control and
test groups. Besides, there were no significant differences between sham and control groups in all tests. Both
anomers led to a significant increase in glutathione, and acetylcholine levels while they caused a decrease in lipid
peroxidation and protein carbonyl levels in brain tissue. It seems that intranasal administration of both anomers
positively influenced maze learning in scopolamine receiving subjects. Although both anomers resulted in similar
biochemistry tests, a higher dose of β anomer indicated better results than
α anomer not only in behavioral tests
but also in biochemical tests.
1. Introduction
(Cummings and Back, 1998; Bowen et al., 1992; Bartus et al., 1982). The
protection or replacement of neurotransmitters, therefore, becomes the
basis for the treatment of AD. Nowadays, the most efficient strategy to
enhance cholinergic transmission is through acetylcholine esterase
(AChE) inhibitors. AChE inhibitors donepezil, rivastigmine, and gal-
antamine are approved for the treatment of AD. These drugs are the first-
line agents in the treatment of AD (Farlow, 2002). The use of AChE in-
hibitors helps in increasing and maintaining the levels of ACh in the
CNS. However, most of these drugs lead to several unwanted side effects.
Hence, there is an urgent need for novel therapeutic molecules with
neuroprotective capacity that could be employed as independent or
adjunctive therapy along with neurotransmitter replacement therapy in
AD. Since neurodegeneration in AD involves multiple pathways,
Alzheimer’s disease (AD) is a progressive neurodegenerative disor-
der and cognitive decline that is the most common form of dementia
affecting elderly people. Although the exact etiologic factor of AD is
unknown, evidence has been mounting that supports the hypothesis that
free radical-induced oxidative damage may play a role in the develop-
ment of AD. AD also characterized by an excess of β amyloid proteins,
lipid peroxides, ventricular enlargement, cortical atrophy, and pro-
gressive degeneration of neurons of the basal forebrain cholinergic
¨
system (Moller and Graeber, 1998; Citron, 2010). According to the
cholinergic hypothesis, memory loss and deterioration of cognitive
functions in AD are attributed to the loss of acetylcholine (ACh)
* Corresponding author.
https://doi.org/10.1016/j.brainres.2021.147517
Received 28 February 2021; Received in revised form 30 April 2021; Accepted 10 May 2021
Available online 13 May 2021
0006-8993/© 2021 Published by Elsevier B.V.

N. Ahmadi et al.
Brain Research 1766 (2021) 147517
including oxidative stress, mitochondrial damage, neuroinflammation,
protein aggregation among others, the therapeutic molecules should be
able to simultaneously target multiple pathways and be non-toxic to
humans at high concentrations. Research evidence has highlighted the
therapeutic potential of dietary polyphenols. Among these, Curcumin,
obtained from the spice turmeric (Curcuma longa) possesses anti-
oxidative, anti-inflammatory (da Costa et al., 2019), anti-carcinogenic,
hypocholesterolemia and wound healing properties (Anand et al.,
2008; Shishodia et al., 2008). Curcumin displays neuroprotective effects
in experimental models of CNS diseases (da Costa et al., 2019; Lim et al.,
2001; Thiyagarajan and Sharma, 2004; Yang et al., 2005). Dietary
supplementation with Curcumin reduces neuroinflammation, astrocytic
proliferation, oxidative stress, and amyloid pathology in the AD model
(Grundman and Delaney, 2002; Ringman et al., 2005; Singh et al.,
2014). Curcumin can scavenge reactive species, prevent protein aggre-
gation and induce neurogenesis in vivo (Xu et al., 2007; Priyadarsini
et al., 2003; Kim et al., 2008), and exhibits neuroprotective properties in
different experimental models of AD (Mythri and Srinivas Bharath,
2012; Mythri et al., 2011). However, in vivo application of Curcumin is
limited due to rapid metabolism and/or biotransformation, systemic
elimination, inefficient cellular uptake, and transport across the
blood–brain barrier (BBB), decreased bioavailability, and stability in the
CNS (Garcea et al., 2004; Pan et al., 1999). To circumvent this, lipo-
somes, and micelles have been employed to increase the bioavailability
of Curcumin (Gupta and Dixit, 2011a, 2011b; Li et al., 2012, 2014; Ray
et al., 2011). Nanoparticle formulations have shown promising results in
vitro, but their efficacy in vivo is limited (Bisht et al., 2007; Tiyaboon-
chai et al., 2007). We tried to synthesize Curcumin-glucosides (CGs) as a
novel synthetic system in which glucose helps to deliver curcumin to the
brain via solution in water. There are a few references on the synthesis of
curcumin glycoside by plant cell suspension cultures (Kaminaga et al.,
2003) and by chemical methods (Masada et al., 2007; Vijayakumar and
Divakar, 2005) but there is no report on the synthesis of fusion reaction
so far. Hence, the present study reports for the first time, curcumin- β
2.1.2. Curcumin tetrahydro α-D-glucoside
Orange amorphous powder, yield: 79%; m.p. = 110–112 ◦C; optical
rotation: [
α
]
D
25 =+30.3◦. (c 0.14 in CHCl3).1HNMR (400 MHz, CDCl3):
δ = 7.63 (d, J = 15.8 Hz, H4), 7.16 (dd, H6), 7.09 (dd, H10),6.96(dd,
H3), 6.46 (t, J = 33.5 Hz,H7), 5.87 (d, J = 24.3 Hz, H11), 5.51 (t, H),
3.94 (d, J = 33.3 Hz, H17), 3.90 (s, H12), 3.06 – 2.87 (m, 2H), 2.81 –
2.58 (m, 2H), 2.19 (d, J = 26.5 Hz, 5H), 1.88 (H, OH). 13C NMR (100
MHz): δ = 182.46(C2,C2′), 149.75(C9,C9′), 144.81(C8,C8′), 140.48(C4,
C4′), 127.37(C5,C5′), 125.84(C3,C3′), 119.41(C6,C6′), 115.27(C7,C7′)
111.91(C10,C10′), 100.59(C11,C11′), 77.52(C15,C15′), 69.76(C12,13,
C12, 13′),69.15(C14,C14′), 61.41(C16,C16′), 58.05(OCH3), 52.64(C1).
2.2. Spatial learning and memory
Curcumin
α and β-D-glucose were investigated for their effect on
spatial memory using the Morris water maze behavioral test. CG’s were
administered with two different doses (12.5 and 25 mg/kg; intranasal).
After the intraperitoneal (IP) injection of scopolamine (1 mg/kg), rats
showed impairment in spatial memory compared to that of the control
group in which there was a significant increase in the time percent that
rats spend in the target quarter to find the hidden platform. As shown in
Fig. 2, the learning process in both the control and donepezil treated
Alzheimer group has been occurring and Q2 presences percent to time
total are significantly higher than the scopolamine received group
(45.7% ± 7.2, 44.5% ±5.4, and 35.7% ± 9.7 respectively, P < 0.01) in
the last day of the trial process. Rat’s groups involve the low and high
doses of curcumin
α and β-D glucosides (23.7%±0.02, 27.6%±3.2,
33.6%±4.5, and 28.6%±4.3) as indicated in Fig. 3 have changed the
learning process in the last day to the first day. This result indicated that
the learning process was impaired in this group. On the test day, rats’
presence in the Q2 quarter (target quadrant) differs again with a similar
pattern as trial days. In other words, on the test day, the presence per-
centages of Alzheimer rats in the Q2 quarter were significantly lower
than the other groups (Fig. 4).
and α-D-glucosides using fusion reaction. We also investigated their ef-
fect on spatial learning and memory in a rat model of the Morris water
maze. Finally, glutathione, protein carbonyl, ACh, lipid peroxide, and
antioxidants level were measured in brain tissue samples in all groups.
2.3. Biochemical assays
2. Results
The mean brain tissue and plasma concentration of all groups include
the low and high doses of curcumin
α and β-D glucosides, scopolamine
2.1. Synthesis of curcumin glucosides (CGs)
(Alzheimer), and controls in rats following glutathione level, antioxi-
dant level, lipid peroxide level, protein carbonyl, and acetylcholine
esterase activity are illustrated. The amount of curcumin glucosides,
scopolamine, and donepezil reaching the brain via intranasal
administration.
The fusion reaction (Marco, 2016) is employed in the synthesis of
curcumin
α and β glucosides. α anomer was an orange-red shiny soft
powder but β anomer was an orange-red turbid hard powder. Both of
them were soluble in water. The experimental yield was 79% for
α
The respective concentration mean of glutathione level in the brain
tissue in the control(sham), Alzheimer, donepezil, the high and low
anomer and 87% for β. Analysis techniques is also shown + 3.5◦ and +
30.3 optical rotation for
α and β respectively. Melting point obtained
doses of
α anomer, the high and low doses of β anomer groups were
110-112℃ for 145–148 for
α and β. We employed NMR spectra for
111.1 ± 47.7, 66.03 ± 15.9, 336.8 ± 12.04, 264.9 ± 50.8, 164.04 ±
24.3, 151.56 ± 17.9, and 276.07 ± 55.7 µM/g tissue(Fig. 5). The anti-
oxidant activity in the brain tissue following in the control (sham),
characterizing these two anomers. You can find with following.
2.1.1. Curcumin tetrahydro β -D-glucoside
Alzheimer, donepezil, the high and low doses of
α anomer, the high and
Orange amorphous powder, yield: 87%; m.p. = 145–148 ◦C; optical
low doses of β anomer groups were 962.5 ± 29.5, 416.6 ± 4.5, 1225.3 ±
52.9, 1241.4 ± 14.3, 963.9, 754.02 ± 15.5, and 739.02 ± 29.8 mM/g
tissue(Fig. 6). The lipid peroxide level that of groups were 18.7 ± 1.4,
39.6 ± 12.1, 36.96 ± 15.6, 34.17 ± 5.9, 27.33 ± 4.9, 37.78 ± 8.02, and
15.46 ± 1.02 µM/g tissue (Fig. 7). The carbonyl protein level for all
groups of aforesaid were 1.09 ± 0.5, 0.92 ± 0.5, 1.09 ± 0.17, 1.3 ± 0.3,
1.12 ± 0.3, 1.04 ± 0.2, and 0.99 ± 0.4 nmol/g tissue (Fig. 8). The
percent of the acetylcholine esterase level is measured in plasma for all
samples were 86.5 ± 12.6, 17.29 ± 4.2, 46.95 ± 13.7, 46.5 ± 5.6,
113.65 ± 2.08, 34.59 ± 3.8, and 13.59 ± 4.04 (Fig. 9). The percent of the
acetylcholine esterase level is calculated in brain tissue for all above
samples were 42.4 ± 12.6, 41.8 ± 4.1, 55.8 ± 13.7, 35.3 ± 5.6, 36.24 ±
2.08, 36.4 ± 3.8, and 45.5 ± 4.04.
rotation: [
α
]
25 =+ 3.5◦ (c 0.1 in CHCl3).1HNMR (400 MHz, CDCl3):
δ = 7.62 (d, JD= 15.7 Hz, H4), 7.15 (d, J = 8.1 Hz, H6), 7.08(d, H10),
6.99 (t, J = 17.6 Hz, H7), 6.51 (d, J = 15.8 Hz, H3),5.88 (d, J = 39.6 Hz,
H11), 5.75 (d, J = 8.3 Hz, 8H), 5.63 (s, 6H), 5.29 (t, J = 9.4 Hz, H1), 3.98
(s, CH3), 3.60 (d, J = 5.9 Hz, H14- H13), 3.94 – 3.78 (m, H12), 3.83 –
3.65 (m, H15), 1.64 (s, OH). 13C NMR (100 MHz): δ = 182.46(C2,C2′),
149.75(C9,C9′), 144.81(C8,C8′), 140.48(C4,C4′), 127.37(C5,C5′),
125.84(C3,C3′), 119.41(C6,C6′), 115.27(C7,C7′) 111.91(C10,C10′),
100.59(C11,C11′), 77.52(C15,C15′), 69.76(C12,13,C12, 13′), 69.15
(C14,C14′), 61.41(C16,C16′), 58.05(OCH3), 52.64(C1).
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Brain Research 1766 (2021) 147517
Fig. 1. Synthetic method of curcumin tetra-o-acetyl
α and β-D-glucopyranose.
Fig. 2. Comparison of control (normal saline), Alzheimer (scopolamine 1 mg/kg) and standard treatment (donepezil 1 mg/kg) groups for spatial memory and
learning using Morris water maze test in trial days. Both control and standard treatment groups differ in the last trial day with the Alzheimer group (scopolamine)
significantly (P < 0.01). Error bars represent mean ± SEM, n = 8.
3. Discussion
least 12 days with 3% of yield (Kaminaga et al., 2003). Hence we tried to
synthesize both anomers ( and β) of the CGs using fusion reaction
α
Curcumin is an insoluble compound in water so researchers have
been tried to improve this problem with different methods such as
coated, esterification and conjugated. In hence, we tried to conjugate
curcumin with glucose by esterification. This work lead to increase
curcumin solubility in water. Previous studies have shown that Curcu-
(Marco, 2016) and characterized by NMR (Figs. 10 and 11). In this re-
action, zinc chloride as a catalyst plays a key role. The reaction was
performed at 130℃ in the presence of the catalyst for 1 hr. The fusion
reaction requires a high temperature, but meanwhile, it has a shorter
reaction time than the other methods. The results showed the method
min
enzymatic synthesis, the yield only reached about 55% of curcumin-β-D-
glucose after 2 h (Masada et al., 2007) and it was 48% for curcumin- -D-
α
and β-D-glucosides could be synthesized by three methods. In the
could improve the yield of the reaction for curcumin
sides to 78% and 87%, respectively.
α and β-D-gluco-
α
In order to investigate the effects of the synthesized compounds in
glucose after 72 h (Vijayakumar and Divakar, 2005). In addition to low
yield, the separation remaining enzyme from the final products is
difficult in this method. The Koenig–Knorr reaction is another way to
prepare curcumin β-glucoside. The yield of the reaction is 95% (Parva-
thy et al., 2009); 64% (Bhaskar Rao et al., 2014), and 51% (Mohri et al.,
2003) but this way needs a lot of chemicals and also a phase transfer
catalyst. Suspension culture was also employed for the synthesis of CGs.
This method is time-consuming and takes a lot of time for synthesis at
reversing scopolamine-induced amnesia, both α and β anomers were
administered intranasal in the low and high doses after IP injection of
scopolamine (1 mg/kg) and data compared to that of the control group
(Normal saline). The findings revealed that both
α and β anomers were
significantly effective in decreasing memory impairment induced by
scopolamine injections, with some differences on the day of their
effectiveness. The percentages of presence in the Q2 quarter, for groups,
received
α anomer showed interesting results (Fig. 3, A). On the second
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Fig. 3. (A and B): Comparison the effects of different
doses (12.5 and 25 mg/kg) of Curcumin Glucosides
(α-D glucoside in 3A and β-D glucoside in 3B) on
scopolamine-induced amnesia using Morris water
maze test in rats. For all groups: error bars represent
Mean ± SEM; and n = 8. 3A: Low dose of α-D gluco-
side showed better results than Alzheimer group on
the second day (P < 0.01) but not on the last day;
which represents state dependent learning rather than
spatial memory trial. 3B: Low dose of β -D glucoside
showed much better results than Alzheimer group on
the last trial day (P < 0.01).
Fig. 4. Comparison of spatial memory among in all groups on test day. CGs were studied for their effect on spatial memory in scopolamine-induced amnesia at two
doses (12.5 and 25 mg/kg; intranasal) except control groups for each anomer with solely the high dose of the anomer. Data are presented as the mean ± SEM. All
groups were compared to Scopolamine received group(P < 0.05).
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Brain Research 1766 (2021) 147517
of the trial process (Fig. 3) whereas, no positive effect on the test day
(Fig. 4). On the other hand, β anomer seems to be effective in spatial
memory impairment caused by scopolamine in the hippocampus,
because of its gradual augmenting effect on learning in trial tests and
spatial memory on the test day (Fig. 4); which in turn needs to be proved
by further histochemical investigations. In comparison with other re-
ports, our data indicated better results than learning memory. They
showed after three-month of treatment with curcumin (da Costa et al.,
2019), rats find platform sooner while with our protocol found platform
just only after 14 days of treatment. Therefore, in this study not only
synthesized components (curcumin glucoside) but also employed
method and protocol seem to be better.
Curcumin has poor oral bioavailability, but the synthesis of CGs
improved this problem. Therefore, using curcumin or nanocarriers such
as NLC (Meng et al., 2015) and metals (Dakhel et al., 2015) for drug
delivery was a good decision. However, reports indicated negligible
effects for them both treatment and prevention (Potter, 2013; Hama-
guchi et al., 2010; Lee et al., 2013; Ahmed and Gilani, 2009). But we
observed the different effects for CGs.
Fig. 5. Comparison of glutathione level in brain tissue for all groups consists of
Control, Alzheimer(ALZ), Donepezil (DON), Alzheimer + Donepezil(Alzheimer
Glutathione level of the brain in AD is decreased as reported in the
literature (Lovell et al., 1998). There is a significant difference between
the Alzheimer group and both of the anomer doses of CGs as well as
donepezil groups (Fig. 5). They increased the level of glutathione in the
test group, but it is inordinate. It could be because of the excess of CGs
and donepezil, which react with DTNB as Michael’s addition and lead to
add intensity of curves.
group receiving Donepezil),
α and β curcumin glucosides(contain control and
Alzheimer groups), Beta(control with 25 mg/kg), Alpha(control with 25 mg/
kg), Alzheimer group receiving β curcumin glucosides(ALZ + β) and Alzheimer
group receiving
α curcumin glucosides (ALZ + α). CGs evaluated in every two
doses (12.5 and 25 mg/kg). The differences in the mean values among the
treatment groups are greater than would be expected; there is a statistically
significant difference (P = <0.001). Error bars represent mean ± SEM; n = 5.
Using of GSH level in the present study practically complicated the
interpretation of results regarding obtaining correct and accurate
reduced glutathione level. To solve this problem, we decided to deter-
mine the FRAP level of the brain. There are many antioxidants in the
brain and plasma, including vitamins, glutathione peroxidase, catalase,
melatonin, and other antioxidants (de Oliveira et al., 2019). They
informed the defense system mechanism of the body against free radi-
cals and oxidative. AD’s and Parkinson’s disease lead to the elimination
of antioxidants in brain tissue and create CNS disorder (Calabrese et al.,
2008). Therefore, measurement of FRAP level is the criteria to assess the
total antioxidant defense level to be able to overcome Michael’s addition
reaction in GSH level. Although in the AD animal model the GSH level
significantly reduced compared to control rats. The study of antioxidants
in the brain tissue showed that the Alzheimer group has the lowest level
of antioxidants (Fig. 6). It can be considered as a verification of the
above note. The data indicated a significant decrease in the FRAP level
in Alzheimer’s rats compared with the control or non-AD groups.
day of the trial process, the low dose
α anomer caused a significant
augmented spatial memory similar to that of state-dependent learning
which followed by a diminished memory again according to what we
can obtain in state-dependent learning memory. However, the high dose
of this anomer did not have any recognizable effect on learning and
memory. The presence percentages of the low and high doses of β
anomer were a bit different on the first and third day (Fig. 3, B) but the
results were similar to
α anomer.
The results of the test day indicated, only the low dose of β anomer
was significantly effective (P < 0.05) in retrieving memory impairment
caused by scopolamine (Fig. 4). According to both trial and test days’
results, it can be deduced that
α anomer in the presence of scopolamine
seems to have some effect on the amygdala and it is effective in the
retrieval of state-dependent learning impairment which can be caused
by scopolamine. Because it showed a very good effect on the second day
Fig. 6. Comparison of antioxidant activity in all
groups such as treatment and control groups. Control,
Alzheimer(ALZ), Donepezil (DON), Alzheimer
Donepezil(Alzheimer group receiving Donepezil),
+
α
and β curcumin glucosides(contain control and Alz-
heimer groups), Beta(control with 25 mg/kg), Alpha
(control with 25 mg/kg), Alzheimer group receiving β
curcumin glucosides(ALZ + β) and Alzheimer group
receiving
α curcumin glucosides (ALZ + α). CGs
evaluated in every two doses (12.5 and 25 mg/kg).
Treatment groups increased antioxidant activity in the
brain in comparison Alzheimer group. There is a sta-
tistically significant difference (*P < 0.05, **P < 0.01,
***P < 0.001). Error bars represent mean ± SEM; n =
5.
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Fig. 7. Comparison of lipid peroxide level in brain tissue for all groups
including Control, Alzheimer(ALZ), Donepezil (DON), Alzheimer + Donepezil
(Alzheimer group receiving Donepezil),
α and β curcumin glucosides(contain
control and Alzheimer groups), Beta(control with 25 mg/kg), Alpha(control
with 25 mg/kg), Alzheimer group receiving β curcumin glucosides(ALZ + β)
and Alzheimer group receiving
α curcumin glucosides (ALZ + α). CGs evaluated
in every two doses (12.5 and 25 mg/kg). Lipid peroxidation occurs in the brain
of rats. Brain homogenates were prepared from the cerebral cortex of rats and
the extent of lipid peroxidation was evaluated by determining the production of
MDA. Data, expressed as micromoles per gram of protein, are means ± SEM of
the values from at least 8 animals. The differences in the mean values among
the treatment groups are greater than would be expected; there is a statistically
significant difference (P = <0.001).
Fig. 9. Comparison of acetylcholine level (A) in plasma, the differences in the
mean values among the treatment groups are greater than would be expected;
there is a statistically significant difference (P = <0.001). (B) In brain tissue,
The differences in the mean values among the treatment groups are not great
enough to exclude the possibility that the difference is due to random sampling
variability; there is not a statistically significant difference (P = 0.332). Control
and treatment groups consist Control, Alzheimer(ALZ), Donepezil (DON), Alz-
heimer + Donepezil(Alzheimer group receiving Donepezil),
α and β curcumin
glucosides(contain control and Alzheimer groups), Beta(control with 25 mg/
kg), Alpha(control with 25 mg/kg), Alzheimer group receiving β curcumin
glucosides(ALZ + β) and Alzheimer group receiving
α curcumin glucosides
(ALZ + α). CGs evaluated in every two doses (12.5 and 25 mg/kg).All treatment
groups have a positive result. Error bars represent mean ± SEM; n = 8.
rats were groups significantly compared with AD rats, but the rise in
FRAP level was at the lowest level compared to other treatments (P >
0.05).
The studies clearly show that involved regions of the brain from
patients with advanced AD have increased levels of lipid peroxidation
products compared to the controls (Montine et al., 2002; Manczak et al.,
2016). According to Fig. 7, the data showed that control and AD rats
were significantly different in MDA level; while in the presence of the
Fig. 8. Comparison of carbonyl protein level in the brain of treatment groups
with Control, Alzheimer(ALZ), Donepezil (DON), Alzheimer + Donepezil(Alz-
heimer group receiving Donepezil),
α and β curcumin glucosides(contain con-
trol and Alzheimer groups), Beta(control with 25 mg/kg), Alpha(control with
25 mg/kg), Alzheimer group receiving β curcumin glucosides(ALZ + β) and
high dose of CGs (α and β anomers) lipid peroxide levels were low.
Alzheimer group receiving
α curcumin glucosides (ALZ + α) . CGs evaluated in
However, none of the treatments were able to reduce the augmented
level of lipid peroxide induced by scopolamine. It is because, according
to the literature, the extent of the lipid peroxidation process cannot be
appraised because the reference values for these parameters are not
known (Jeandel et al., 1989) and Carbonyl (CO) groups (aldehydes and
ketones) are produced in protein side chains (especially of Pro, Arg, Lys,
and Thr) when they are oxidized.
every two doses (12.5 and 25 mg/kg). decreased carbonyl protein level, The
differences in the mean values among the treatment groups are not great
enough to exclude the possibility that the difference is due to random sampling
variability; there is not a statistically significant difference (P = 0.141). Error
bars represent mean ± SEM; n = 8.
Furthermore, no significant difference was observed in FRAP in all
treated groups compared with control rats. As shown in Fig. 6, FRAP
Protein carbonyl derivatives can also be generated through oxidative
cleavage of proteins by either α-amidation pathway or by oxidation of
levels in Donepezil and α-anomer -treated groups increased compared
glutamyl side chains (Berlett and Stadtman, 1997). Interestingly,
with AD rats. However, increase their FRAP levels of β anomer treated
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Fig. 10. A) 1H NMR (400 MHZ, CDCl3) spectra of Curcumin tetrahydro β-D-glucoside. B) 13C NMR (100MHZ, CDCl3) spectra of Curcumin tetrahydro β-D-glucoside.
increased concentrations of protein carbonyls have been documented in
normal aging as well as in AD and Parkinson’s disease. That is why we
measured the level of carbonyl protein in all groups (Fig. 8). The Alz-
heimer group has a high level of carbonyl protein while the rest of the
group reduced its level. Although β anomer doses decreased CO group
level more than the control group, and Donepezil and doses of
did it as less as the control group.
α-anomer
Acetylcholine esterase (AChE) level in the brain tissue decreased in
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Brain Research 1766 (2021) 147517
Fig. 11. A) 1HNMR (400 MHZ, CDCl3) spectra of Curcumin tetrahydro
α
-D-glucoside. B) 13CNMR (100 MHZ, CDCl3) spectra of Curcumin tetrahydro
α-D-glucoside.
Alzheimer’s Disease (Lombardo and Maskos, 2015). There is a correla-
tion between the loss of ACh and decline in mental status which leads to
the cholinergic hypothesis of cognitive impairment in AD. As shown in
Fig. 9, the results confirmed the ACh level of the control group is more
than the Alzheimer group either at plasma or the brain. All of the
treatments increased ACh levels in plasma and brain tissue except
anomer in the case of brain tissue. In plasma, donepezil, the low and
high doses of anomer augmented significantly ACh level into the low
α
α
8

N. Ahmadi et al.
Brain Research 1766 (2021) 147517
dose of β anomer. It was able to increase ACh level in the Alzheimer
group. None of the groups expanded on the ACh level to the control
group level. In the brain tissue, donepezil and both doses of β anomer
swim and find the platform, during which the time to find the hidden
platform (escape latency) was measured. At the end of each run, rats
were wiped and put under heather. Spatial memory retention on the test
day, can be evaluated by the software on the basis of the time, which
animals spent within the “target” quadrant which contains the hidden
platform during training days.
increased ACh level, despite
α anomer which even declined ACh level.
So donepezil and β anomer performed successfully at plasma and brain
tissue.
In conclusion, the present study confirms a novel synthesis method of
CGs in good yields with shorter reaction time and in simple and eco-
friendly reaction conditions. The results supported the increased hy-
drophilicity and biological activity of CGs. The data also reveal the
compounds CGs showing enhanced antioxidant and activities against
4.3.2. Plasma and brain collection
On the last day (10th day), all rats were anesthetized using the CO2
chamber, blood samples were taken and their plasma was separated.
Simultaneously, the brain tissues were quickly isolated in an ice - bath
and prepared for biochemical examinations.
AD. It was observed that the CGs containing both anomer
α and β were
significantly increased glutathione and ACh level in vivo and decreased
LPO and carbonyl protein in the animal models. These results uphold the
fact that CGs in comparison with donepezil as a commercial drug have
potential in reducing the progress of AD with less side effect. Our find-
ings also support other results that have been reported about the pro-
tective and therapeutic effect of curcumin and its derivatives in AD in
the literature (Lim et al., 2001; Ahmed and Gilani, 2009; Brondino et al.,
2014; Aggarwal and Harikumar, 2009).
4.3.3. Antioxidant assay
100 mg of each brain tissue sample was homogenized in 1 ml of
Trichloroacetic acid (TCA, 6%) and centrifuged at 10000 rpm. Then, 1.5
ml of ferric reducing Antioxidant power (FRAP) reagent was added into
the supernatant and the absorbance was recorded at 593 nm.
4.3.4. Assay of lipid peroxidation level
Lipid peroxidation (LPO) was measured by determining malo-
nyldialdehyde (MDA) and using thiobarbitoric acid (TBA) which has
been used as an indicator of lipid peroxidation (Ohkawa et al., 1979;
Kandimalla et al., 2016). 100 mg of each brain tissue sample was ho-
mogenized in 1 ml of KCl (0.156 M) and centrifuged at 3500 rpm, the
supernatant was removed and 2.5 ml sulfuric acid (0.05 M), and 2 ml
TBA (0.2% w/w) was added to precipitate. It was put in a boiling water
bath for 30 m, cooled, and then 4 ml of n-butanol was added to the
mixture. It was centrifuged at 3500 rpm and the absorbance at 532 nm
(Mahadik et al., 1995) was recorded.
4. Materials and methods
4.1. Drugs and reagents
Curcumin, glucose, and scopolamine were purchased from Out Co.
(Germany), Merck Co. (Germany), and Sigma- Aldrich Co. (America)
respectively. Donepezil was a gift of Sobhan Daro, Rasht, Iran. All other
chemicals in this study, which are analytical-reagent grade, were pur-
chased locally from the Amertat Shimi pharmaceutical Co. (Mamuniyeh,
Iran) and Ezmiran Co. (Tehran, Iran).
4.3.5. Assay of glutathione level.
Reduced glutathione (GSH) as a non-enzymatic intracellular anti-
oxidant defense protects cells from the damaging effects of ROS level. It
also maintains the thiol-containing proteins in their active form.
Briefly,100 mg of each brain tissue sample was homogenized in 1 ml of
EDTA (0.02 M), then 1.5 ml of TCA (10% w/w) was added and centri-
fuged. 2.5 ml of Tris buffer (pH = 8.9) and 0.5 ml of DTNB (5, 5′-
dithiobis-2-nitrobenzoic acid) as GSH reagent was added to 1 ml of su-
pernatant. The change in the absorbance at 412 nm was followed and
the GSH level was represented as µg mg -1 protein.
4.2. Synthesis of curcumin glucosides (CGs)
4.2.1. Synthesis of curcumin tetrahydro β-D-glucoside and α-D-glucoside
A solution of zinc chloride in anhydride acetic (14 ml) was stirred in
an oil bath at 130℃ while Penta acetylꢀ β-D-glucopyranose (1.12 g, 2.9
mmol) and curcumin (0.48 g, 1.3 mmol) were added slowly. After an
hour, the solution was washed with cold water and cold saturated
NaHCO3 solution, dried over MgSO4 and concentrated to leave the tetra
acetyl-β-D-glucoside curcuminas an orange-red solid (Fig. 1). NaOH:
MeOH (10 ml) was added after cooling to room temperature, and the
mixture was stirred for about 1hr (until the solid dissolved) and ester
hydrolyzed to form β-D-glucoside curcumin. The same method was
4.3.6. Assay of acetylcholine esterase activity
Inhibition of AChE activity was determined by a modified micro
assay method of Ellman (Zhang et al., 2014). The enzyme hydrolyzes the
substrate acetylthiocholine and results in the product thiocholine which
reacts with Elman’s reagent (DTNB) to produce 2-nitrobenzoate-5-
mercaptothiocholine and 5-thio-2-nitrobenzoate which can be detec-
ted at 412 nm. According to the procedure, 40 µL of AChE (75 mm in
buffer with pH 8) and 10 µL of the sample solution were added to 3.0 ml
of pH 8 buffer and pre-incubated on ice (4℃) for 30 min. Duplicate tubes
(blank control and test groups of plasma) were also treated in the same
way. The reaction was triggered by adding 10 µL of 0.25 mM DTNB and
10 µL of 3 mM ACh, and the solution was incubated at 37℃ for 20 min.
Optical density was measured at 412 nm immediately and inhibition
percentage was calculated. We measured AChE activity in the brain
tissue samples according to the same method just to add 1 ml of Hya-
mine after step incubated at 37℃. All the measurements were carried
out in triplicate.
employed for the synthesis of α-D-glucoside curcumin.
4.3. Animals
100 Wistar male rats weighed 180–200 g were bought from Razi
Institute (Tehran, Iran). They were divided into 11groups. Animals
housed in the social condition under a 12hr light: dark cycle at 21–23 ◦C
and were allowed access to food and water ad libitum. All procedures
were in accordance with the National Institutes of Health (NIH) Guide
for the Care and Use of Laboratory Animals. It was also approved by the
Animal Ethics Committee of Zanjan University of Medical Sciences. The
ethical approval code was: ZUMS. REC.1396.228. All the handlings were
performed during the light phase and between 10 a.m. to 16p.m.
4.3.1. Behavioral assessment
Morris water maze (MWM) behavioral test is used to evaluate the
spatial learning memory as described fully by Anoush, et al, in 2020, and
in literature (Kandimalla et al., 2018; Manczak et al., 2018). Animals
were trained for 3 consecutive days in the initial learning phase, started
from day 7 of drug administration, in order to learn the place of the
platform and try to find it. For each trial, rats were placed in the pool at
one of four quadrants (chosen randomly by the software) and allowed to
4.4. Statistical analysis
All data are presented as mean ± SEM values. All data were analyzed
by one-way analysis of variance (ANOVA), followed by Tukey’s post hoc
test. A probability (P) value of <0.05 was considered significant. Sta-
tistical analysis was performed using SPSS 23.0 software package for
9

N. Ahmadi et al.
Brain Research 1766 (2021) 147517
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Hamaguchi, T., Ono, K., Yamada, M., 2010. Curcumin and Alzheimer’s disease. CNS
Neurosci. Ther. 16, 285–297.
CRediT authorship contribution statement
Jeandel, C., Nicolas, M., Dubois, F., Nabet-Belleville, F., Penin, F., Cuny, G., 1989. Lipid
peroxidation and free radical scavengers in Alzheimer’s disease. Gerontology 35,
275–282.
Nahid Ahmadi: Conceptualization, Writing - original draft, Soft-
ware. Mir-Jamal Hosseini: Visualization, Data curation. Kobra Ros-
tamizadeh: Supervision. Mahdieh Anoush: Visualization, Data
curation, Writing - review & editing.
Kaminaga, Y., Nagatsu, A., Akiyama, T., Sugimoto, N., Yamazaki, T., Maitani, T.,
Mizukami, H., 2003. Production of unnatural glucosides of curcumin with drastically
enhanced water solubility by cell suspension cultures of Catharanthus roseus. FEBS
Lett. 555, 311–316.
Kandimalla, R., Manczak, M., Fry, D., Suneetha, Y., Sesaki, H., Reddy, P.H., 2016.
Reduced dynamin-related protein 1 protects against phosphorylated Tau-induced
mitochondrial dysfunction and synaptic damage in Alzheimer’s disease. Hum. Mol.
Genet. 25, 4881–4897.
Declaration of Competing Interest
Kandimalla, R., Manczak, M., Yin, X., Wang, R., Reddy, P.H., 2018. Hippocampal
phosphorylated tau induced cognitive decline, dendritic spine loss and
mitochondrial abnormalities in a mouse model of Alzheimer’s disease. Hum. Mol.
Genet. 27, 30–40.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Kim, S.J., Son, T.G., Park, H.R., Park, M., Kim, M.-S., Kim, H.S., Chung, H.Y., Mattson, M.
P., Lee, J., 2008. Curcumin stimulates proliferation of embryonic neural progenitor
cells and neurogenesis in the adult hippocampus. J. Biol. Chem.
Lee, W.-H., Loo, C.-Y., Bebawy, M., Luk, F., Mason, R.S., Rohanizadeh, R., 2013.
Curcumin and its derivatives: their application in neuropharmacology and
neuroscience in the 21st century. Curr. Neuropharmacol. 11, 338–378.
Li, C., Zhang, Y., Su, T., Feng, L., Long, Y., Chen, Z., 2012. Silica-coated flexible
liposomes as a nanohybrid delivery system for enhanced oral bioavailability of
curcumin. Int. J. Nanomed. 7, 5995.
Acknowledgments
This work was supported by the school of pharmacy, Zanjan Uni-
versity of medical sciences.
Li, H., Zhang, N., Hao, Y., Wang, Y., Jia, S., Zhang, H., Zhang, Y., Zhang, Z., 2014.
Formulation of curcumin delivery with functionalized single-walled carbon
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