Curcumin Monoglucoside Shows Improved Bioavailability and Mitigates Rotenone Induced Neurotoxicity in Cell and Drosophila Models of Parkinson’s Disease

Article abstract of DOI:10.1007/s11064-016-2034-6

Curcumin (CUR), a dietary polyphenol has diverse pharmacologic effects, but is limited by poor bioavailability. This is probably due to decreased solubility, cellular uptake and stability. In order to enhance its solubility and bioavailability, we synthesized the CUR bioconjugate curcumin monoglucoside (CMG) and tested its bioavailability, neuroprotective and anti-apoptotic propensity against rotenone (ROT) induced toxicity in N27 dopaminergic neuronal cells and Drosophila models. Our results elucidate that CMG showed improved bioavailability than CUR in N27 cells. Pre-treatment with CMG protected against ROT neurotoxicity and exerted antioxidant effects by replenishing cellular glutathione levels and significantly decreasing reactive species. CMG pre-treatment also restored mitochondrial complex I and IV activities inhibited by ROT. ROT-induced nuclear damage was also restored by CMG as confirmed by comet assay. CMG induced anti-apoptotic effects was substantiated by decreased phosporylation of JNK3 and c-jun, which in turn decreased the cleavage of pro-caspase 3. Q-PCR analysis of redox genes showed up-regulation of NOS2 and down-regulation of NQO1 upon ROT exposure and this was attenuated by CMG pre-treatment. Studies in the Drosophila ROT model revealed that, CMG administration showed better survival rate and locomotor activity, improved antioxidant activity and dopamine content than ROT treated group and was comparable with the CUR group. Based on these data, we surmise that CMG has improved bioavailability and offered neuroprotection comparable with CUR, against ROT-induced toxicity both in dopaminergic neuronal cell line and Drosophila models, with therapeutic implications for PD.

Full text of DOI:10.1007/s11064-016-2034-6

Neurochem Res
DOI 10.1007/s11064-016-2034-6
ORIGINAL PAPER
Curcumin Monoglucoside Shows Improved Bioavailability
and Mitigates Rotenone Induced Neurotoxicity in Cell
and Drosophila Models of Parkinson’s Disease
M. D. Pandareesh1,2 · M. K. Shrivash3 · H. N. Naveen Kumar4 · K. Misra3 ·
M. M. Srinivas Bharath1,2
Received: 15 May 2016 / Revised: 7 August 2016 / Accepted: 10 August 2016
© Springer Science+Business Media New York 2016
Abstract Curcumin (CUR), a dietary polyphenol has
diverse pharmacologic effects, but is limited by poor
bioavailability. This is probably due to decreased solu-
bility, cellular uptake and stability. In order to enhance
its solubility and bioavailability, we synthesized the
CUR bioconjugate curcumin monoglucoside (CMG)
and tested its bioavailability, neuroprotective and anti-
apoptotic propensity against rotenone (ROT) induced
toxicity in N27 dopaminergic neuronal cells and Dro-
sophila models. Our results elucidate that CMG showed
improved bioavailability than CUR in N27 cells. Pre-
treatment with CMG protected against ROT neurotox-
icity and exerted antioxidant effects by replenishing
cellular glutathione levels and significantly decreas-
ing reactive species. CMG pre-treatment also restored
mitochondrial complex I and IV activities inhibited by
ROT. ROT-induced nuclear damage was also restored by
CMG as confirmed by comet assay. CMG induced anti-
apoptotic effects was substantiated by decreased phos-
porylation of JNK3 and c-jun, which in turn decreased
the cleavage of pro-caspase 3. Q-PCR analysis of redox
genes showed up-regulation of NOS2 and down-regula-
tion of NQO1 upon ROT exposure and this was attenu-
ated by CMG pre-treatment. Studies in the Drosophila
ROT model revealed that, CMG administration showed
better survival rate and locomotor activity, improved
antioxidant activity and dopamine content than ROT
treated group and was comparable with the CUR group.
Based on these data, we surmise that CMG has improved
bioavailability and offered neuroprotection comparable
with CUR, against ROT-induced toxicity both in dopa-
minergic neuronal cell line and Drosophila models, with
therapeutic implications for PD.
M. D. Pandareesh and M. K. Shrivash have contributed equally to
this work.
Electronic Supplementary Material The online version of this
article (doi:10.1007/s11064-016-2034-6) contains supplementary
material, which is available to authorized users.
ꢀ M. M. Srinivas Bharath
Keywords Curcumin · Rotenone · Neuroprotection ·
Antioxidant · Bioavailability · Parkinson’s disease ·
Drosophila melanogaster
bharath@nimhans.ac.in; thathachar@rediffmail.com
1
Department of Neurochemistry, National Institute of Mental
Health and Neurosciences, No. 2900, Hosur Road,
Bangalore 560029, Karnataka, India
Abbreviations
2
CMG Curcumin monoglucoside
ROS Reactive oxygen species
GSH Glutathione (reduced)
Neurotoxicology Laboratory at the Neurobiology
Research Center, National Institute of Mental Health and
Neurosciences, No. 2900, Hosur Road, Bangalore 560029,
Karnataka, India
CI
CIV
PD
Mitochondrial complex I
Mitochondrial complex IV
Parkinson’s disease
3
Department of Chemistry, Centre of Bio-Medical Research
(CBMR), Sanjay Gandhi Post Graduate Institute of Medical
Sciences Campus, Raebareli Road, Lucknow 226014,
Uttar Pradesh, India
JNK c-Jun N-terminal kinase.
4
Department of Biochemistry, Jnana Sahyadri, Kuvempu
University, Shankargatta 577451, Karnataka, India
1 3

2
Neurochem Res
Introduction
neuroprotective effects of bioconjugates of CUR against
oxidative/nitrosative stress in cell models [14, 34].
Parkinson’s disease (PD) is an age-related progressive
neurodegenerative disorder characterized by the selective
loss of substantia nigra (SN) dopaminergic neurons lead-
ing to depletion of dopamine in the striatum [1]. Conven-
tional pharmacotherapies employed to treat PD patients
replenish striatal dopamine and provide symptomatic relief.
However, most of these drugs do not have the capacity to
either halt or slow down the neurodegenerative process in
the SN. Hence, there is an urgent need for novel therapeu-
tic molecules with neuroprotective capacity that could be
employed as independent or adjunctive therapy along with
dopamine replacement therapy in PD. Since neurodegenera-
tion in PD involves multiple pathways 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 evidences have highlighted the therapeutic
potential of dietary polyphenols. Among these, Curcumin
(CUR) (diferuloylmethane), obtained from the spice tur-
meric (Curcuma longa) possesses anti-oxidative, anti-
inflammatory, anti-carcinogenic, hypocholesterolemic
and wound healing properties [2, 3]. CUR displays neuro-
protective effects in experimental models of CNS diseases
[4–6]. Dietary supplementation with CUR reduces neu-
roinflammation, astrocytic proliferation, oxidative stress
and amyloid pathology in the Alzheimer’s disease (AD)
model [4, 7–9]. CUR can scavenge reactive species, pre-
vent protein aggregation and induce neurogenesis in vivo
[10–12] and exhibits neuroprotective properties in differ-
ent experimental models of PD [13, 14]. CUR mitigates
varied therapeutic effects through suppression of NF-κB/
IκB pathway, c-jun N-terminal kinase, cyclooxygenase 2,
Bcl-2 and Bcl-xL and cellular inhibitor of apoptosis pro-
tein-1 [15].
However, in vivo application of CUR is limited due to
rapid metabolism and/or biotransformation, systemic elimi-
nation, inefficient cellular uptake and transport across the
blood brain barrier (BBB), decreased bioavailability and
stability in the CNS [13, 16, 17]. To circumvent this, lipo-
somes, and micelles have been employed to increase the
bioavailability of CUR [18–23]. Nanoparticle formulations
have shown promising results in vitro, but their efficacy in
vivo is limited [24, 25]. Small molecular-weight surfactants,
such as cetyltrimethylammonium bromide [26–28], syn-
thetic amphiphilic polymers, such as poly(ethylene oxide)-
b-poly(caprolactone) [29] and methoxy poly(ethylene
glycol)-palmitate [30], have also been employed to encap-
sulate CUR. CUR treatment as a phospholipid complex
[31], or in combination with piperine improved its bioavail-
ability within the brain [32, 33]. We previously tested the
Most of the approaches have attempted to improve the
solubility of CUR moiety for easier cellular uptake. In this
study, we synthesized curcumin monoglucoside (CMG),
by linking the hydroxyl group of glucose to one of the
phenoxyl groups of CUR to facilitate easier uptake into cells
and improved bioavailability. We have also evaluated the
neuroprotective property of CMG against rotenone (ROT)
induced toxicity in N27 cell and Drosophila models.
Materials and Methods
All chemicals and solvents were of analytical grade. Bulk
chemicals and solvents were obtained from Merck and
Sisco Research Laboratories Pvt. Ltd. (Mumbai, India).
Fine chemicals including HPLC consumables, cell cul-
ture consumables, and α-dinitrophenyl (DNP) antibody
were obtained from Sigma (Sigma Aldrich, India). Fetal
calf serum for cell culture was obtained from PAN Bio-
tech (GmBh, Germany). Antibodies to JNK, phospho-JNK,
c-jun, phospho-c-jun, caspase-3 were obtained from Abcam
(Abcam, MA, USA). Horseradish peroxidase (HRP) conju-
gated secondary antibodies were obtained from Bangalore
Genei. Nitrocellulose membrane was obtained from Milli-
pore (Millipore, Billerica, MA, USA).
Synthesis of CMG
The synthesis of CMG was carried out step-wise as follows:
Preparation of 1,2,3,4,6-pentacetyl-β-d-glucopyranose
Amixture of d-glucose (50.0 g, 0.277 mol), acetic anhydride
(283 g, 2.77 mol) and sodium acetate (30.0 g, 0.366 mol)
were incubated with continuous stirring at 100°C for 5 h.
The reaction was monitored as described [35] and the prod-
uct was crystallized from ethanol as white powder (89 g;
yield, 82%; mp. 130°C).
Preparation of 2,3,4,6-tetra-O-acetyl
1-bromo-d-glucopyranose
To the solution of 1,2,3,4,6-pentacetyl-β-d-glucopyranose
(60.0 g, 0.15 mol) in glacial acetic acid (120 ml), hydrobro-
mic acid (150 ml, 33%) was added. The reaction mixture
was incubated with continuous stirring at 5°C for 30 min fol-
lowed by incubation at room temperature for 5 h. The com-
pletion of reaction was monitored as described [36] and the
crude product 2,3,4,6-tetra-O-acetyl 1-bromo-d-glucopyra-
nose was crystallized from diethyl ether/petroleum ether as
white crystalline solid (58.70 g; yield, 92%; mp. 86–88°C).
1 3

Neurochem Res
3
Synthesis of CUR
101.61, 100.17, 72.37, 71.97, 70.99, 68.19, 61.79, 55.99,
20.66, 20.58, 20.53.
A mixture of vaniline (60.8 g; 0.4 mol), boric acid (24 g;
0.38 mol), m-xylene (13.2 g), acetylacetone (20.8 g;
0.21 mol) and n-butyl amine (1.9 ml; 0.02 mol) were dis-
solved in dimethyl formamide (70 ml) and refluxed in
dean stark apparatus under reduce pressure (18 mm Hg)
at 58–62°C for 2 h. The mixture of n-butyl amine (0.8 ml;
0.01 mol) in DMF (20 ml) was added to the reaction mix-
ture and refluxed for 2 h. The completion of reaction was
monitored with TLC. The reaction mixture was diluted with
mixture of acetic acid (110 ml) and water (20 ml). This solu-
tion was slowly added to the round bottom flask containing
540 ml water and 60 ml acetic acid stirred at 80°C for 2 h,
cooled to room temperature and filtered, washed with cold
water, and the crude product crystallized from methanol as
yellow crystals (55 g; yield, 71%; mp.182–183°C) [37].
Preparation of 4-O-(β-d-glucosyl) CUR
To the solution of 4-O-(acetylated glucosyl) CUR (5 g;
0.007 mol) in dry methanol (100 ml), sodium methoxide
(1.9 g; 0.03 mol) was added and the solution stirred for 30 min
at 0°C. The completion of reaction was monitored with TLC.
The reaction mixture was neutralized with Dovex H+ resin.
The resin was filtered and the filtrate was concentrated under
vacuum, at 20°C. The crude product was purified on column
chromatography, eluted with DCM: methanol (85:15v/v)
and the orange coloured crystalline product was obtained
1
(3.5 g yield, 92%; mp. 110–111°C). H NMR (800 MHz,
Methanol-d4) δ 7.62–7.55 (m, 1H), 7.30–7.16 (m, 3H), 7.14–
7.10 (m, 1H), 6.83 (d, J=8.2 Hz, 1H), 6.73–6.62 (m, 1H), 4.99
(d, J=7.6 Hz, 1H), 3.96–3.86 (m, 6H), 3.76–3.40 (m, 5H),
3.32 (p, J=1.6 Hz, 3H). 13C NMR (201 MHz, Methanol-d4)
δ 184.26, 182.29, 149.56, 149.14, 148.44, 148.00, 141.13,
139.72, 129.93, 127.08, 122.83, 122.38, 122.06, 120.86,
115.94, 115.16, 110.91, 110.29, 100.76, 76.88, 76.42, 73.39,
69.86, 61.05, 55.31, 55.02.
Synthesis of 4-O- and 4, 4′-di-O-(2,3,4,6-tetra-O-acetyl-d-
glucosyl) CUR
To the stirring solution of CUR (4.08 g; 0.011 mol) in
dichloromethane (75 ml), aqueous solution of KOH (75 ml,
15%) was added followed by addition of benzyl tetra butyl
ammonium chloride (3.45 g; 0.011 mol), 2,3,4,6-tetra-
O-acetyl-1-bromo-d-glucopyranose (11.5 g; 0.02 mol) in
dichloromethane (50 ml) and the reaction mixture was stirred
for 5h at room temperature (25 1°C).The completion of
reaction was monitored by TLC. The organic layer was sep-
arated and washed with 50 ml (0.1 N) NaOH solution, brine
solution, and water, dried over sodium sulphate and concen-
trated under high vacuum. The product thus obtained was a
mixture of CUR (1 g), 4-O-(acetylated glucosyl)-CUR (4a)
yield 2.5 g (25%), mp. 86–88°C and 4,4′-di-O-(acetylated
glucosyl) CUR (4b) with yield 5 g (44%), mp. 172–174°C.
The mixture was separated on silica gel column and eluted
with ethyl acetate:hexane (40:60 v/v) gradient [38, 39].
(4a) 4-O-(acetylated glucosyl)CUR: 1H NMR (800 MHz,
Chloroform-d) δ 7.59 (d, J=15.7 Hz, 1H), 7.18–7.03 (m,
3H), 6.53 (d, J=15.7 Hz, 1H), 5.36–4.97 (m, 4H), 4.29 (dd,
J=12.3, 5.0 Hz, 1H), 4.18 (dd, J=12.3, 2.5 Hz, 1H), 4.01–
3.68 (m, 5H), 2.21–1.97 (m, 15H). 13C NMR (201 MHz,
Chloroform-d) δ 183.06, 170.57, 170.25, 169.39, 169.31,
150.67, 147.63, 139.96, 131.45, 123.34, 121.56, 119.48,
111.45, 101.64, 100.22, 72.40, 72.00, 71.01, 68.21, 61.81,
56.02, 20.69, 20.61, 20.57.
Cell Culture and Cell Viability
The N27 dopaminergic cell line used throughout the study
was grown and maintained as described [40]. The cells were
treated with freshly prepared ROT (prepared in DMSO and
subsequently diluted in culture media) for 24 h with or with-
out pretreatment with CMG and CUR for 24 h. Cell viability
was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) assay [40] and LDH release
assay [41]. After the treatment period, 10 µl of cell lysis
solution (2% Triton X-100) was added to the untreated cells
to obtain 100% cell death.
Bioavailability of CMG and CUR in N27 Cells
Levels of CMG or CUR were estimated using high perfor-
mance liquid chromatography (HPLC) with UV/Vis detector
as described [42]. In brief, the cells treated with and without
CMG or CUR were homogenized in 500 µl of ice cold sodium
phosphate buffer (0.1 M; pH 6.8) containing 0.1% EDTA and
centrifuged at 2500×g for 10 min at 4°C. The supernatant was
sequentially extracted with methanol, hexane and ethyl acetate
fraction. CMG or CUR was concentrated by removing ethyl
acetate by vacuum evaporation and the residue was recon-
stituted in methanol. Twenty microliter of this sample was
injectedvia7725irheodyneinjectorintoaSunfire® C18column
(4.6×250 mm, 5 μm ID) (Waters, India) of an HPLC system
(Waters, India) to separate the curcuminoids. The mobile phase
consisting of 50 mM potassium dihydrogen orthophosphate
1
(4b) 4,4′ –di-O- (acetylated glucosyl) CUR: H NMR
(800 MHz, Chloroform-d) δ 7.59 (d, J=15.8 Hz, 1H), 7.17–
7.02 (m, 5H), 6.53 (d, J=15.7 Hz, 1H), 5.84 (s, 1H), 5.37–
4.97 (m, 7H), 4.24 (dd, J=70.0, 3.8 Hz, 3H), 3.87 (s, 6H),
2.22–1.90 (m, 24H).13C NMR (201 MHz, Chloroform-d)
δ 183.04, 175.20, 170.57, 170.24, 169.38, 169.31, 150.63,
147.60, 139.94, 131.41, 123.30, 121.54, 119.41, 111.43,
1 3

4
Neurochem Res
(pH adjusted to 3.5 using orthophosphoric acid): acetonitrile
(40:60 v/v) was pumped isocratically at a flow rate of 0.8 ml/
min. The detection was carried out using a UV–Visible detec-
tor at 424 nm. The retention time of CMG was 3.65 min while
the retention time of bisdemethoxy CUR, demethoxy CUR
and CUR were 8.59, 9.25 and 10.02 min respectively. The
Empower™ 3 software was utilized for integration.
X-100 and 10% DMSO; pH 10.0) for 120 min in dark at 4°C
to lyse the cellular and nuclear membranes. The slides were
rinsed with unwinding buffer (3 M NaOH, 10 mM EDTA;
pH 13.0) and transferred into an electrophoresis tank contain-
ing unwinding buffer for denaturing the DNA, followed by
electrophoresis for 30 min at 25 V. The slides were washed
twice with neutralizing buffer (0.4 M Tris-HCl; pH 7.5) for
10 min and treated with ethanol for another 5 min. The slides
were stained with 40 µl of ethidium bromide (10 mg/ml) and
DNA damage was visualized by using fluorescence micro-
scope (Olympus, Japan equipped with Cool SNAP® Pro color
digital camera). The damage appeared as a ‘comet’with frag-
mented DNA (tail) being separated from undamaged nuclear
DNA (head) and measurements were made by Comet Assay
IV software to determine the olive tail movement (%). The
results were expressed as percent tail movement.
Redox Assays
Cellular reactive oxygen species were estimated using the
oxidation-sensitive dye 2′,7′-dichlorfluorescein-diacetate
as described [43]. Total cellular hydroperoxides were mea-
sured using commercially available amplex red kit (A22188,
Invitrogen) as per the manufacturer’s instructions.
Lipid peroxidation was estimated by thiobarbituric acid
reactive substances method [44] with minor modifications.
The malondialdehyde (MDA) content was calculated using
a molar extinction coefficient of 1.56×105 M−1 cm−1 [45].
Protein oxidation was estimated by oxyblots as described
earlier [43] using the dinitrophenyl hydrazine method with
minor modifications [46]. Western signals were densito-
metrically quantified and normalized against the respective
anti-tubulin signal.
Mitochondrial Assays
CI enzyme assay was carried out as previously described
[47, 50] and the results were plotted as relative ROT-
sensitive specific activity. CIV activity was measured by
recording the decrease in absorbance due to the oxidation of
reduced cytochrome c at 550 nm as described [14].
Total glutathione level was estimated by the 5, 5′dithiobis
2-nitrobenzoic acid recycling method [47, 48]. The GSH con-
tent was calculated based on the maximum reaction rate com-
pared with GSSG standards and normalized per mg protein.
Total RNA Isolation, c-DNA Synthesis
and Semi-quantitative PCR
Total RNA was extracted from N27 cells using RNeasy spin
columns (Qiagen, USA) as per the manufacturer’s instruc-
tions. cDNA was synthesized using commercially available
kit from Applied Biosystems (4368814) (CA, USA) accord-
ing to the manufacturer’s instructions, and stored at −20°C.
Transcript abundance of nitric oxide synthase 2 (NOS2)
and NAD(P)H:quinone oxidoreductase 1 (NQO1) genes were
examined by quantitative PCR. Primers were designed using
Integrated DNA technologies (IDT) and primer-BLAST soft-
ware (Table 1). GAPDH was used as housekeeping gene.
The PCR reaction mix contained 2 ng of total cDNA, 2X eva
green ready mix (containing dNTPs,10 µl Taq-Polymerase)
and 10 pmol forward and reverse primers (Sigma–Aldrich)
Single Cell Gel Electrophoresis (SCGE) Assay
Apoptosis was measured by quantitating DNA damage by
alkaline comet assay [49] with minor modifications. N27 cells
(1×106 cells) seeded in 100 mm petri plate and subjected to
various treatments were collected and mixed with 0.5% (w/v)
low melting agarose (LMA) in 0.01 M phosphate buffered
saline. The mixture was pipetted onto frosted slides pre-coated
with 1% (w/v) normal melting agarose. After solidification of
agarose, the slides were covered with 100 µl of 0.5% (w/v)
LMA and immersed in lysis buffer (2.5 M NaCl, 100 mM
EDTA, 10 mM Tris-HCl buffer, 0.1% SDS and 1% Triton
Table 1 Primers sequences used for quantitative RT-PCR analysis
Gene
Accession no.
Primers
Position of the primers
in the cDNA
Product size (bp)
GAPDH
NOS2
NM_017008
NM_012611
NM_017000
Forward–5′ GGTGCTGAGTATGTCGTGGA 3′
Reverse–5′ CGGAGATGATGACCCTTTTG 3′
Forward–5′ GCTACTACTCCATCAGCTCCTC 3′
Reverse–5′ GTCTTCGGGCTTCAGGTTATTG 3′
Forward–5′ TCAGGTGGCCTGGGATATG
Reverse–5′ CAACAAGTGGGTGGAGGATTG
340–359
417–436
97
149
140
2898–2919
3025–3046
1091–1109
1210–1230
NQO1
1 3

Neurochem Res
5
in a final volume of 20 µl. The cycling programs were as
follows: 10 min of reverse transcription at 50°C, one cycle
and 5 min of polymerase activation at 95°C followed by 45
cycles of PCR at 95°C for 10 s, 58°C for 30 s. This was fol-
lowed by melt curve analysis at 65–95°C with 1% increment
for 10 s to verify the authenticity of the amplified product by
its specific melting temperature (Tm) using the melting curve
analysis software (Life-technologies 7500 software v2.0.6).
The threshold cycle (Ct) of the gene of interest, housekeeping
gene and the difference between their Ct values (∆Ct) were
determined. Normalized gene expression was calculated
using Life-technologies 7500 software v2.0.6 [51].
one end. After a brief recovery period, flies were gently tapped
to the bottom of the column. After 1 min, the number of flies
that reached the top of the column and flies that remained at
the bottom were counted separately. Data was expressed as
percent flies escaped beyond minimum distance of 10 cm in
60 s [54, 55]. The score for each replicate was an average of
three such trials for each experimental group including control.
Redox Assays
Head and rest of the body from 25 flies were separated and
homogenized in sodium-phosphate buffer (0.1 M; pH 7.4)
followed by centrifugation (2500×g for 10 min at 4°C). The
supernatant was used directly to estimate ROS, hydroper-
oxide and reduced glutathione (GSH) levels as described in
the previous section.
Western Blot Analysis
Total cellular protein was separated on SDS-PAGE and
transferred onto a nitrocellulose membrane using an electro
blotting apparatus (Bio-Rad, Hercules, CA, USA) [52]. The
membranes were probed with primary antibodies against
β-actin (ab8227), c-jun (ab32137), p-c-jun (ab32385), JNK
(55 A8), p JNK (98F2), pro and active caspase 3 (ab13585)
(Abcam, MA, USA) (all at 1:1000) followed by horseradish
peroxidase conjugated secondary antibodies (goat anti-rabbit
and anti-mouse antibodies) (Millipore, Billerica, MA, USA)
(at 1:10,000). The membranes were developed using chemilu-
minescence, its intensity captured using advanced gel doc sys-
tems (G:BOXChemi XT4, Syngene, MD, USA) and the band
intensity was measured using NIH imageJ analysis software.
Estimation of Dopamine (DA) and its Metabolites by
HPLC
Levels of DA, 3,4-Dihydroxyphenylacetic acid (DOPAC)
and homovanillic acid (HVA) were estimated using HPLC
with electrochemical detector as described [56]. In brief, the
head/rest of the body of 60 flies were homogenized in ice
cold 0.4 M perchloric acid containing 5 mM sodium bisul-
fite and 0.04 mM EDTA to avoid oxidation. Homogenates
were centrifuged (33,000×g for 15 min at 4°C) and 20 µl of
the supernatant was injected via HPLC pump (Model 1525,
Waters, USA) into a column (Sunfire®, RP C18, 5 μm par-
ticle size, 4.6×100 mm at 30°C) connected to an electro-
chemical detector (Model 2465, Waters, USA) at a potential
of +650 mV with glassy carbon working electrode versus
Ag/AgCl reference electrode. Mobile phase consisted of
0.125 M sodium citrate buffer containing 20% methanol,
0.1 mM Na2EDTA, 0.5 mM 1-octanesulfonic acid sodium
salt adjusted to pH 4.3. The flow rate was set at 1.0 ml/
min. DA, DOPAC and HVA were identified and quantified
by comparing their retention time and peak area to those of
respective standards and expressed in ng/mg protein.
Drosophila melanogaster Experiments
Drosophila Husbandry and Treatment
Synchronized D. melanogaster, wild (Oregon K), adult
male flies (10 days old) were maintained at 24 1°C and
70–80% relative humidity, and fed on a standard wheat
flour-agar diet as described [53].
Apilot study was carried out by exposing the flies to ROT,
CUR and CMG at 10, 100, 500 and 1000 µM for 14 days
to determine lethality. For neuroprotective studies of CMG
and CUR, only one concentration of ROT (ED50; 500 µM)
(prepared in DMSO) was employed. Briefly, the flies were
pretreated with either CMG or CUR via diet for 5 days. Sixth
day onwards, flies were exposed to ROT for the next 7 days.
The flies were then subjected to negative geotaxis and later
sacrificed by exposing to −80°C for 3 min. Drosophila head
was separated from the rest of the body using a sharp cutter
and stored in −80°C until further analysis.
Determination of Protein
Protein concentrations in N27 cells and Drosophila head
and body homogenates were determined by the Bradford
method [57] using bovine serum albumin as standard.
Statistical Analysis
The data were expressed as mean standard deviation of the
mean (SEM). Data were analyzed using one-way ANOVA
followed by Tukey’s post hoc test using SPSS 15.0 for Win-
dows software. Differences at p<0.05 were considered to
be significant.
Negative Geotaxis Assay
Twenty adult male flies were anesthetized and placed in a ver-
tical glass column (length, 25 cm; diameter, 1.5 cm) sealed at
1 3

6
Neurochem Res
Scheme 1
Stepwise synthesis of Curcumin monoglucoside (CMG)
OH
O
OAc
O
Acetic anhydride
HO
HO
AcO
AcO
Sodium acetate
100 0C, 5h
OH
OH
OAc
OAc
1
OAc
O
OAc
O
HBr in acetic acid
AcO
AcO
AcO
AcO
0 0C,0.5h
RT,5h
Br
OAc
OAc
OAc
2
OAc
O
O
OH
O
OH
AcO
AcO
Br
O
O
OAc
O
O
KOH, BTBACl,
H2O,DCM,RT,
5h
R1O
OR2
HO
OH
3
4a,R1 =H,R2=Tetraacetyl glucose
4b,R1=R2= Tetraacetyl glucose
O
OH
O
OH
O
O
O
O
MeOH,NaOMe
0.5h, 00C
O
OH
O
OH
OH
OAc
Dovex H+
O
5
O
OH
OH
OAc
OAc
HO
CMG
AcO
4a
Fig. 1 Scheme of organic synthesis of CMG. The glucose moiety was
synthesized as pentacetate β-d-glucose and then converted to aceto-
bromoglucose (1, 2). CUR (3) was treated with acetobromoglucose
(2) to obtain a mixture of 4-O- and 4, 4′-di-O-(2,3,4,6-tetra-O-acetyl-
d-glucosyl) CUR (4a, 4b). The 4-O-(2,3,4,6-tetra-O-acetyl-d-gluco-
syl) CUR (4a) was deacetylated to obtain 4-O-(β-d-glucosyl) CUR (5)
Results
was deacetylated to obtain 4-O-(β-d-glucosyl) CUR (5 in the
scheme), with 92% yield and was characterized by 1H NMR
and 13C spectra (Supplementary Fig. 1).
Synthesis and Characterization of CMG
The scheme of synthesis of CMG is shown in Fig. 1. CUR
synthesized with vaniline and acetylacetone [37] was used
to generate CMG. The glucose moiety was synthesized as
pentacetate β-d-glucose and then converted to acetobromo-
glucose (1 and 2 in the scheme). CUR (3 in the scheme) was
treated with acetobromoglucose to obtain a mixture of 4-O-
and 4, 4′-di-O-(2,3,4,6-tetra-O-acetyl-d-glucosyl) CUR (4a
and 4b in the scheme). These two glucosides were separated
Cytotoxicity of ROT and Protective Effect of CMG
Against Cell Death
Treatment of N27 cells with ROT (10–2000 nM) for 24 h
caused neuronal death in a dose-dependent manner (Fig. 2a).
Treatment with 500 nM ROT for 24 h caused 50% cell death
and the same concentration was used for future experiments.
Cells pre-treated with CMG or CUR (0.25−5 µM) for 24 h
were significantly protected against ROT (500 nM) at 5 µM
of CMG and 1 µM of CUR (Fig. 2b, c). These results were
on silica gel column and characterized by 1H NMR and 13
C
spectra. The 4-O-(2,3,4,6-tetra-O-acetyl-d-glucosyl)-CUR
1 3

Neurochem Res
7
A
B
125
125
100
75
50
25
0
ROT (500 nM)
100
75
50
25
0
#
#
*
#
*
#
*
#
*
*
*
*
*
CON 10 25 50 100 250 500 1000 2000
Rotenone (nM)
CON
0
0.25
0.5
1
2.5
5
CMG ( M)
C
D
ROT (500 nM)
125
500
400
300
200
100
0
*
*
100
75
50
25
0
#
#
#
#
*
#
*
*
*
*
*
*
CON 10
25
50
100 250 500 1000 2000 TX100
CON
0
0.25
0.5
1
2.5
5
CUR ( M)
Rotenone (nM)
E
300
250
200
150
100
50
*
#
#
0
CON
CMG
ROT
CMG+ROT CUR+ROT
Fig. 2 CMG protects against ROT-dependent cell death in N27 cells.
a Dose-dependent effect of ROT on N27 cell viability. b, c Shows
dose dependent protective effect of CMG and CUR on ROT induced
cytotoxicity in N27 cells, respectively. d Dose-dependent effects of
ROT on LDH release. e Protective effect of CMG and CUR on ROT
(500 nM) induced LDH release in N27 cells. The data are represented
as mean SEM of three independent experiments. *p<0.05 versus
control group and #p<0.05 versus ROT treated group
50
45
40
35
further confirmed by LDH release assay supporting the neu-
roprotective efficacy of CMG compared to CUR (Fig. 2d, e).
Bioavailability of CMG Levels Within N27 Cells
30
5
Next, we compared the bioavailability of CMG and CUR
in N27 dopaminergic neuronal cells. N27 cells were treated
with CMG and CUR (5 µM each) at different time inter-
vals (0–24 h) followed by quantitation of their intra cellular
levels (Fig. 3). At 30 min time-point, the concentration of
CMG and CUR were 38.72 0.99 and 2.46 0.12 pg/mg
of protein respectively. The maximum level of CMG was
found to be 42.92 1.25 pg/mg protein after 3 h of treat-
ment. While CUR levels drastically decreased after 18 h of
treatment, CMG concentration was found to be 35.32 0.93
CMG
CUR
4
3
2
1
0
0.5
1
3
6
12
18
24
Time (h)
Fig. 3 Bioavailability of CMG and CUR in N27 cells at different time
intervals (0–24 h). The data are represented as mean SEM of three
independent experiments
1 3

8
Neurochem Res
D
CMG
CUR
A
800
600
400
200
0
*
ROS
#
#
CON
CMG
ROT
CMG+ROT CUR+ROT
B
500
Hydroperoxides
300
250
200
150
100
50
*
*
400
300
200
100
0
#
#
#
#
0
CON
CMG
ROT
CMG+ROT CUR+ROT
CON
CMG
ROT
CMG+ROT CUR+ROT
30
25
20
15
10
5
40
30
20
10
0
C
E
*
#
#
#
#
*
0
CON
CMG
ROT
CMG+ROT CUR+ROT
CON
CMG
ROT
CMG+ROT CUR+ROT
Fig. 4 CMG displays antioxidant effect against ROT-dependent oxi-
dative damage in N27 cells. Protective effects of CMG against ROT
induced a ROS production b hydroperoxide levels c lipid peroxidation
d protein oxidation and e total GSH levels are shown; AU arbitrary
units. The data are represented as mean SEM of three independent
experiments. *p<0.05 versus control group and #p<0.05 versus ROT
treated group
and 32.80 1.77 after 18 and 24 h of treatment respectively,
indicating its increased transport across the cell membrane
and availability in N27 cells compared to CUR (Fig. 3).
hydroperoxides were significantly scavenged (p<0.05) by
CMG (119.62 and 256.27%) and CUR (116.78 and 215.47%)
pre-treatment respectively, indicating comparable antioxidant
propensity of CMG compared to CUR (Fig. 4a, b).
CMG Displays Antioxidant Activity and Protects
Mitochondrial Function in N27 Cells
N27 cells exposed to ROT (500 nM) for 24 h showed
increased lipid peroxidation, as indicated by MDA levels,
which was elevated by 219.71% compared to control. This
was significantly decreased following pre-treatment with
CMG (158.44%) and CUR (157.25%) (p<0.05) (Fig. 4c).
Similarly ROT-dependent protein oxidation (262.94% com-
pared to control) was significantly decreased by pre-treat-
ment with CMG (184.49%) and CUR (143.89%) (Fig. 4d).
Since CUR offers neuroprotection primarily due to its antioxi-
dant property [58], we tested whether CMG has similar anti-
oxidant capacity as CUR. ROT treatment (500 nM) elicited
1.75 and 4.18 fold increase in ROS and hydroperoxides levels,
respectively, in N27 cells, compared with control. ROS and
1 3

Neurochem Res
9
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Effect of CMG on Apoptotic Markers
*
CON
CMG
ROT
CMG+ROT
ROT-dependent apoptotic markers were evident in N27 cells
as indicated by DNA damage. SCGE assay revealed that
ROT treated cells displayed definite tail (21.39%) with con-
sistent amount of DNA fragments, indicating genomic dam-
age (Fig. 7a). CMG pre-treatment significantly decreased
the tail movement by 10.43% (Fig. 7b), indicating protec-
tive efficacy of CMG against ROT-induced DNA damage.
Next we tested the status of apoptotic proteins relevant to
ROT neurotoxicity and PD. Accordingly, ROT treatment acti-
vated JNK by increased phosphorylation of JNK (240.5%),
c-jun (133.33%) and subsequent cleavage of procaspase-3
(261.6%). Pre-treatment with CMG significantly restored the
expression of all these apoptotic protein markers to the control
level indicating the anti-apoptotic role of CMG (Fig. 7c–e).
#
#
*
NOS2
NQO1
Fig. 5 CMG modulates the expression of Redox genes in the ROT cell
model. Quantitative PCR data show that CMG restored the expression
of the redox genes NOS2 and NQO1 gene altered by ROT in N27
cells. The data are represented as mean SEM of three independent
experiments. *p<0.05 versus control group and #p<0.05 versus ROT
treated group
ROT-induced oxidative damage was associated with
decreased level of total glutathione (41.24% compared to
control), and this was significantly restored following pre-
treatment with CMG (67.06%) and CUR (86.68%). These
data indicate that CMG displayed antioxidant activity, but
this was slightly lower than CUR (Fig. 4e).
Protective Effect of CMG in the ROT Fly Model
Exposure of Drosophila melanogaster adult male flies
(n=60) to ROT (10–1000 µM) showed dose-dependent
mortality. ROT at 1000 µM induced detectable mortality on
day 3, while exposure for 14 days showed 100% mortality.
ROT exposure at 500 µM for 6 days induced ~50% mortality
(Fig. 8a). On the other hand, both CMG and CUR treated flies
were non-toxic up to day-6 and day-7 respectively (Fig. 8b,
c). Pre-treatment of flies for 6 days with CMG (500 µM) or
CUR (500 µM) against ROT treatment (500 µM) markedly
reduced the incidence of mortality (Fig. 8d). ROT treated
flies exhibited ~70% locomotor impairments as indicated by
the negative geotaxis assay, while pre-treatment with either
CMG or CUR significantly alleviated locomotor deficits in a
dose dependent manner (Fig. 8e).
To further investigate the redox dynamics, we analyzed
the expression of nitric oxide synthase2 (NOS2) [59] and
NAD(P)H:quinone oxidoreductase 1 (NQO1) genes [60,
61], which are biomarkers of oxidative stress and antioxi-
dant defense respectively. Quantitative RT-PCR showed
ROT-induced over-expression of NOS2 by 3.43 fold while
the NQO1 gene was under-expressed by 0.29 fold compared
to control. Upon pre-treatment with CMG, expression levels
of both the genes were restored to control level (Fig. 5).
Altered redox dynamics induced by ROT is associated
with mitochondria dysfunction [62]. Accordingly, the activ-
ities of mitochondrial complexes I and IV were declined by
ROT exposure to 56.96 and 73.56% respectively, compared
to control, while pre-treatment with CMG and CUR signifi-
cantly restored both complex I (77.64 and 79.02% respec-
tively) and complex IV (88.98 and 95.43% respectively)
activities respectively (Fig. 6a, b).
Effect of CMG on Oxidative Stress Markers in the
ROT Fly Model
FliesexposedtoROTtreatmentexhibitedmarkedlyincreased
ROS (body region: 279.30 17.45% and head region:
158.84 7.96%) and hydroperoxides (269.75 4.83%
A
B
150
200
#
#
150
#
#
*
100
50
0
100
50
0
*
CON
CMG
ROT
CMG+ROT CUR+ROT
CON
CMG
ROT
CMG+ROT CUR+ROT
Fig. 6 CMG restores mitochondria function in the ROT cell model.
Protective effect of CMG against ROT induced complex I (a) and com-
plex IV activity (b) is shown. The data are represented as mean SEM
of three independent experiments. *p<0.05 versus control group and
#p<0.05 versus ROT treated group
1 3

10
Neurochem Res
CMG
CON
ROT
A
B
i
ii
c
25
20
15
10
5
*
ROT
*
#
CMG+ROT
iii
iv
0
CON
CMG
CMG+ROT
C
CMG
ROT
p JNK3
(54 &46 kDa)
300
200
100
0
#
JNK3
(54 kDa)
Actin
(42 kDa)
CON
CMG
ROT
CMG+ROT
D
CMG
ROT
150
*
#
p c-jun
100
50
0
(42 kDa)
c-jun
(40 kDa)
CON
CMG
ROT
CMG+ROT
Actin
(42 kDa)
300
250
200
150
100
50
E
*
CMG
ROT
#
Procaspase 3
( 32 kDa)
Active
Caspase 3
( 14-21 kDa)
0
CON
CMG
ROT
CMG+ROT
Actin
(42 kDa)
1 3

Neurochem Res
11
Fig. 7 CMG restores the apoptotic markers induced by ROT in N27
cells. a shows the protective effect of CMG on DNA damage induced
by ROT in N27 cells. i Control cells without treatment, ii CMG (5 µM),
iii ROT (0.5 µM), iv cells pre-treated with CMG (5 µM) for 24 h and
followed by ROT (0.5 µM). b Quantitation of tail movement in the
COMET assay shown as %. c–e CMG restores the level of apoptotic
markers proteins-phospho c-jun, phospho JNK and caspase 3 induced
by ROT, as analyzed by western blot. Quantitation of the individual
bands in each blot is indicated in the bar graph adjacent to the blot. The
data are represented as mean SEM of three independent experiments.
*p<0.05 versus control group and #p<0.05 versus ROT treated group
compared to control flies (31.23 1.99 ng/mg protein and
12.65 0.68 ng/mg protein). Pre-treatment with CMG and
CUR effectively scavenged the free radicals induced by
ROT treatment, in the fly body and head (p>0.05) and also
significantly restored the GSH content (Table 2).

Neuromodulatory Effect of CMG Against ROT
Induced Dopamine (DA) Depletion
Flies exposed to ROT showed significantly decreased DA
(66.76 7.78 ng/mg protein), DOPAC (51.85 3.75 ng/
mg protein) and HVA (26.54 2.14 ng/mg protein) lev-
els compared to control group (DA=205.85 6.88 ng/
and 154.27 3.60%) levels compared to control group
(100%) (Table 2). These were associated with GSH deple-
tion (14.34 1.21 and 6.89 0.30%) in ROT treated flies
CMG
ROT
A
B
60
60
40
20
0
CON
VEHICLE CON
CMG10
CMG100
CMG500
CMG1000
CON
VEHICLE CON
ROT10
ROT100
ROT500
ROT1000
50
40
30
30
0
1
2
3
4
5 6 7 8 9 10 11 12 13 14
1
2
3
4
5
6
7 8 9 10 11 12 13 14
Days
Days
CUR
D100
C
60
50
40
CON
CMG
CUR
ROT
CMG+ROT
CUR+ROT
CON
VEHICLE CON
CUR10
CUR100
CUR500
CUR1000
80
60
30
30
0
40
40
0
1
2
3
4
5
6
7 8 9 10 11 12 13 14
6
7
8
9
Days
10
11
12
Days
E
150
100
50
#
#
#
#
#
#
#
#
#
#
*
0
CON DMSO ROT 50
100 250 500 1000
50
100 250 500 1000
CMG (µM)
CUR (µM)
Fig. 8 CMG improves ROT-induced mortality in the fly model of
PD. Dose dependent lethality response expressed as percent mortality
among adult male Drosophila melanogaster exposed to various con-
centration of a ROT, b CMG, c CUR is shown. d Shows % mortality
among adult male flies pretrea in vivo thereby enhancing the antioxi-
dant potentialted with CMG or CUR for 5 days. On day-6 onwards
flies were exposed to ROT in combination with CMG or CUR. e
Restoration of ROT induced locomotor deficits by CMG and CUR,
as determined by negative geotaxis assay. The data represented as
mean SEM of three independent experiments. *p<0.05 versus con-
trol group and #p<0.05 versus ROT treated group
1 3

12
Neurochem Res
mg protein, DOPAC=71.69 4.50 ng/mg protein and
HVA =39.36 3.54 ng/mg protein), while flies exposed to
CMG or CUR alone did not alter the levels of DA and its
metabolites (Fig. 9a–c). On the other hand, pre-treatment
with CMG and CUR prevented ROT-dependent DA deple-
tion (99.25 8.67 and 94.25 9.21 ng/mg protein respec-
tively). CMG and CUR effectively reversed the elevated
ratio of (DOPAC+HVA) to DA induced by ROT treatment
suggesting that CMG and CUR pre-treatment inhibited the
turnover of DA (Fig. 9d).
Discussion
Although CUR displays numerous health benefits, its low
bioavailability due to low water solubility and rapid metab-
olism has been the major impediment in its in vivo applica-
tion. Phase I clinical trials have shown that CUR exhibited
poor oral bioavailability [6]. Upon CUR metabolism, it
undergoes sulfatation, methylation and glucuronidation and
these conjugated metabolites can be found in plasma after
CUR injection [63]. These metabolites show reduced bioac-
tivity and will be excreted.
Consequently, enhancement of CUR bioavailability
would be of utmost importance in order to exert therapeu-
tic effects, in vivo. Various attempts made to improve the
bioavailability of CUR include improved intestinal absorp-
tion via absorption enhancers [18], novel delivery systems
such as CUR nanoparticles, liposomal CUR or CUR phos-
pholipid complexes [64] and improving metabolic stability
via adjuvants such as piperine to prevent glucuronidation
[65]. In the current study, we synthesized CUR bioconju-
gates by blocking the phenoxyl group with glucose, through
biodegradable ester bonds. Glycosylation improves the
bioavailability by better solubility due to increased hydro-
philicity and enhanced metabolic stability due to protection
of phenolic hydroxyl groups. Previous study on CMG has
investigated its effect on the in vitro aggregation of alpha-
synuclein [66]. However, a comprehensive analysis of the
neurpoprotective properties of CMG in a cell and fly model
has not been conducted so far.
Both mono and di-O-glucosides (CMG and CDG) of CUR
were prepared in the current study. This approach could delay
the metabolic degradation of CUR along with the accumula-
tion of CUR and the desired ligand in the cell followed by the
action of esterases [34, 67]. It is well known that the antioxi-
dant activity of CUR is mainly due to its free phenolics [13].
In silico docking of CMG and CDG to the two most impor-
tant molecular targets of PD viz. α-synuclein and cAbI indi-
cated higher binding energy for CDG as compared to CMG
and CUR (Data not shown). However our wet experiments
showed that CMG is more active than CDG (Data not shown).
This can be explained by hypothesizing that although CDG is
1 3

Neurochem Res
13
A
B
250
100
75
50
25
0
200
150
100
50
*
#
#
#
#
*
0
CON
CMG
CUR
ROT
ROT+CMG ROT+CUR
CON
CMG
CUR
ROT
ROT+CMG ROT+CUR
C
D
50
40
30
20
10
0
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00
*
*
#
#
#
#
CON
CMG
CUR
ROT ROT+CMGROT+CUR
CON
CMG
CUR
ROT
ROT+CMG ROT+CUR
Fig. 9 Effect of CMG on DA and its metabolites in N27 cells. Quanti-
tation of a DA, b DOPAC and c HVA in different experimental groups
is shown. The (DOPAC+HVA)/DA ratio is shown in (d). The data are
represented as mean SD of three independent experiments. *p<0.05
versus control (CON) group and #p<0.05 versus ROT treated group
more hydrophilic than CMG, both phenolics are blocked in
the former and not readily available to scavenge free radicals
necessary for the antioxidant property of CUR.
antioxidant activity of CMG is lower than CUR since one
of the phenoxyl groups is covalently bonded to a glycosyl
moiety (Fig. 4). In our earlier studies we have shown that
CUR induces GSH synthesis by activation of GCL activity
in vivo thereby enhancing the antioxidant potential [34, 73].
We found that CMG effectively restored the GSH levels
depleted by ROT treatment (Fig. 4e). Based on these data,
we propose that the improved bioavailability of CMG made
up for lower antioxidant capacity compared to CUR.
ROS plays a major role in initiating apoptosis, [74] which
can be inhibited by improved antioxidants levels [75]. Our
data showed ROT dependent apoptosis in N27 cell was
indicated by genomic damage and the same was signifi-
cantly mitigated by CMG (Fig. 7a). CUR targets c-jun N
terminal Kinase (JNK) pathway preventing JNK1/2 and
c-jun phosphorylation and caspase 3 activation [76]. CUR
does not directly inhibit kinase activity of JNK [77] but
probably interferes with the proximal upstream and not the
distal upstream of mitogen activated kinase kinase kinase
level. The inhibition of the upstream activators might not
affect JNK cascade [78]. CUR induces JNK pathway by
inducing anti-apoptotic genes such as Bcl2 and inhibits
iNOS resulting in reduction of ROS and oxidative stress
mediated cytochrome c release and caspase 3 activation.
The current study showed that CMG inhibited caspase 3
activation by preventing phosphorylation of JNK and c-jun
(Fig. 7c–e).
According to literature reports [39], the water solubil-
ity of curcumin monoglucoside (CMG) is 230 times higher
than CUR. These results are in support with glycosylation
of other compounds like genistein, where genistein-7-O-α-
d-glucopyranoside showed fourfold increase in solubility
(358 μM) at 25°C, compared with genistein (90 μM) [68].
In the present study, we have shown the CMG bioavailabil-
ity within N27 cells was ~tenfold more compared to CUR.
CMG levels remained high even after 24 h of treatment
whereas CUR levels were below the detectable limits after
18 h of treatment (Fig. 3). Apart from increased hydrophi-
licity and enhanced metabolic stability due to protection
of phenoxyl groups, the cellular uptake could be enhanced
since CMG could be taken up via glucose transporters.
Many investigators showed that ROT, a mitochondrial
respiratory chain complex I inhibitor, could induce cell
death in a variety of cells [58, 69–71]. We observed that ROT
induced cell death (Fig. 2) which was mediated by decreased
complex I activity (Fig. 6) and increased oxidative stress
(Fig. 4) [69, 72]. Our results showed that CMG and CUR
displayed free radical scavenging property (Fig. 4), consis-
tent with our previous studies [34]. We presume that CMG
pre-treatment induced elevated glutathione levels thereby
protecting CI activity against ROT toxicity. These findings
are consistent with our previous study which showed that
CUR pretreatment restored CI activity [14]. However, the
In the current study, we focused on two representative
genes of antioxidant defense (NQO1) and oxidative stress
1 3

14
Neurochem Res
References
(NOS2). NQO1, a homodimeric antioxidant flavoenzyme
contributes to the maintenance of cellular redox state and
protein degradation and catalyzes the reduction of quinones
and aromatic nitro compounds and prevents ROS accumula-
tion [79, 80]. NQO1 is cytoprotective in response to oxida-
tive stress [60, 61] and proteasomal degradation [81, 82].
NQO1 also regulates the cellular NADH/NAD+ratio [83]
and ensuing cell viability [84]. We observed that while ROT
exposure down-regulated NQO1, pretreatment with CMG
restored NQO1 expression (Fig. 5). On the other hand,
NOS2 is an oxidative/nitrative stress marker [85, 86], which
mediates degenerative processes following brain injury [87]
and cerebral ischemia [88]. ROT is known to induce oxida-
tive stress via up-regulation of NOS2 expression [59]. We
observed that while ROT exposure up-regulated NOS2,
pretreatment with CMG restored NOS2 expression (Fig. 5).
Our in vivo study in the fly model showed that ROT
exposure induced dose dependent mortality and locomotor
deficits consistent with previous studies [89]. ROT-induced
neurotoxicity is attributed to the specific sensitivity of dopa-
minergic neurons to mitochondrial dysfunction and ROS
[90]. CMG and CUR treatment decreased ROT-dependent
mortality and locomotor deficits, thus substantiating the
protective property of CMG similar to CUR (Fig. 8). These
findings were consistent with earlier studies which showed
consistent correlation between locomotor dysfunction and
dopamine deficiency [91]. Accordingly, CMG treatment
significantly ameliorated the ROT induced deficits includ-
ing restoration of DA metabolites (Fig. 9) and antioxidant
activity (Table 2). Our study also demonstrates that the ROT
cell and fly models provides a valuable system for preclini-
cal investigation on PD therapeutics.
To summarize, our study demonstrates that CMG, a novel
bioconjugate has better bioavailability than CUR and protects
against ROT induced toxicity in vitro and in vivo via decreas-
ing ROS level, improving intracellular GSH, preventing
dopamine depletion and inhibiting JNK mediated - caspase
3 dependent apoptosis. The advantage of CMG is that while
one free phenoxyl group of CUR contributes to its antioxidant
activity, the glucoside moiety attached to the other phenolic
helps in enhancing solubility, cellular uptake and bioavail-
ability. According to our knowledge, this is the first report
to show that CMG has a protective effect in ROT-based PD
models, suggesting that CMG could offer better pharmacoki-
netics and pharmacodynamics compared to CUR in PD.
1. Mercado G, Valdes P, Hetz C (2013) An ERcentric view of Par-
kinson’s disease. Trends Mol Med 19:165–175
2. Anand P, Thomas SG, Kunnumakkara AB et al (2008) Biologi-
cal activities of curcumin and its analogues (Congeners) made by
man and mother nature. Biochem Pharmacol 76:1590–1611
3. Shishodia S, Misra K, Aggarwal B (2008) Turmeric as cure-
cumin: promises, problems, and solutions. In: Dietary modulation
of cell signaling pathways. Taylor & Francis, Inc., p 91
4. Lim GP, Chu T, Yang F et al (2001) The curry spice curcumin
reduces oxidative damage and amyloid pathology in anAlzheimer
transgenic mouse. J Neurosci 21:8370–8377
5. Thiyagarajan M, Sharma SS (2004) Neuroprotective effect of
curcumin in middle cerebral artery occlusion induced focal cere-
bral ischemia in rats. Life Sci 74:969–985
6. Yang F, Lim GP, Begum AN et al (2005) Curcumin inhibits for-
mation of amyloid beta oligomers and fibrils, binds plaques, and
reduces amyloid in vivo. J Biol Chem 280:5892–5901
7. Grundman M, Grundman M, Delaney P (2002) Antioxidant strat-
egies for Alzheimer’s disease. Proc Nutr Soc 61:191–202
8. Ringman JM, Frautschy SA, Cole GM et al (2005) A potential
role of the curry spice curcumin in Alzheimer’s disease. Curr
Alzheimer Res 2:131–136
9. Singh D, Gupta M, Kesharwani R et al (2014) Molecular drug
targets and therapies for Alzheimer’s disease. Transl Neurosci
5:203–217
10. Kim SJ, Son TG, Park HR et al (2008) Curcumin stimulates pro-
liferation of embryonic neural progenitor cells and neurogenesis
in the adult hippocampus. J Biol Chem 283:14497–14505
11. Priyadarsini KI, Maity DK, Naik GH et al (2003) Role of phe-
nolic O–H and methylene hydrogen on the free radical reactions
and antioxidant activity of curcumin. Free Radical Biol Med
35:475–484
12. Xu Y, Ku B, Cui L et al (2007) Curcumin reverses impaired hippo-
campal neurogenesis and increases serotonin receptor 1 A mRNA
and brain-derived neurotrophic factor expression in chronically
stressed rats. Brain Res 1162:9–18
13. Mythri RB, Bharath MM (2012) Curcumin: a potential neuropro-
tective agent in Parkinson’s disease. Curr Pharm Des 18:91–99
14. Mythri RB, Harish G, Dubey SK et al (2011) Glutamoyl diester
of the dietary polyphenol curcumin offers improved protection
against peroxynitrite-mediated nitrosative stress and damage of
brain mitochondria in vitro: implications for Parkinson’s disease.
Mol Cell Biochem 347:135–143
15. Sharma RA, GescherAJ, Steward WP (2005) Curcumin: the story
so far. Eur J Cancer 41:1955–1968
16. Garcea G, Jones DJ, Singh R et al (2004) Detection of curcumin
and its metabolites in hepatic tissue and portal blood of patients
following oral administration. Br J Cancer 90:1011–1015
17. Pan MH, Huang, Lin JK (1999) Biotransformation of curcumin
through reduction and glucuronidation in mice. Drug Metab Dis-
pos 27:486–494
18. Anand P, Kunnumakkara AB, Newman RA et al (2007) Bio-
availability of curcumin: problems and promises. Mol Pharm
4:807–818
19. Gupta NK, Dixit VK (2011) Development and evaluation of
vesicular system for curcumin delivery. Arch Dermatol Res
303:89–101
20. Gupta NK, Dixit VK (2011) Bioavailability enhancement of cur-
cumin by complexation with phosphatidyl choline. J Pharm Sci
100:1987–1995
Acknowledgments This study was supported by a research grant
from the Department of Biotechnology (DBT), India (Grant No. BT/
PR4908/MED/30/745/2012).
Compliance with Ethical Standards
21. Li C, Zhang Y, Su T et al (2012) Silica-coated flexible liposomes
as a nanohybrid delivery system for enhanced oral bioavailability
of curcumin. Int J Nanomedicine 7:5995–6002
Conflict of Interest The authors declare that there are no conflicts
of interest.
1 3

Neurochem Res
15
22. Li H, Zhang N, Hao Y et al (2014) Formulation of curcumin
delivery with functionalized single-walled carbon nanotubes:
characteristics and anticancer effects in vitro. Drug Deliv
21:379–387
23. Ray B, Bisht S, Maitra A et al (2011) Neuroprotective and neu-
rorescue effects of a novel polymeric nanoparticle formulation
of curcumin (NanoCurc) in the neuronal cell culture and animal
model: implications for Alzheimer’s disease. J Alzheimers Dis
23:61–77
42. Asai A, Miyazawa T (2000) Occurrence of orally administered
curcuminoid as glucuronide and glucuronide/sulfate conjugates
in rat plasma. Life Sci 67:2785–2793
43. Ramadasan-Nair R, Gayathri N, Mishra S et al (2014) Mitochon-
drial alterations and oxidative stress in an acute transient mouse
model of muscle degeneration: implications for muscular dystro-
phy and related muscle pathologies. J Biol Chem 289:485–509
44. Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxidation
in animal tissues by thiobarbituric acid reaction. Anal BioChem-
istry 95:351–358
24. Bisht S, Feldmann G, Soni S et al (2007) Polymeric nanoparticle-
encapsulated curcumin (“nanocurcumin”): a novel strategy for
human cancer therapy. J Nanobiotechnol 5:3
45. Buege JA, Aust SD (1978) Microsomal lipid peroxidation. Meth-
ods Enzymol 52:302–310
25. Tiyaboonchai W, Tungpradit W, Plianbangchang P (2007) For-
mulation and characterization of curcuminoids loaded solid lipid
nanoparticles. Int J Pharm 337:299–306
26. Iwunze MO, McEwan D (2004) Peroxynitrite interaction with
curcumin solubilized in ethanolic solution. Cell Mol Biol (Noisy-
le-grand) 50: 749–752
27. Leung MH, Colangelo H, Kee TW (2008) Encapsulation of cur-
cumin in cationic micelles suppresses alkaline hydrolysis. Lang-
muir 24:5672–5675
28. Wang F, Wu X, Wang F et al (2006) The sensitive fluorimetric
method for the determination of curcumin using the enhancement
of mixed micelle. J Fluoresc 16:53–59
29. Ma Z, HaddadiA, Molavi O et al (2008) Micelles of poly(ethylene
oxide)-b-poly(epsilon-caprolactone) as vehicles for the solu-
bilization, stabilization, and controlled delivery of curcumin. J
Biomed Mater Res A 86:300–310
30. Sahu A, Bora U, Kasoju N et al (2008) Synthesis of novel bio-
degradable and self-assembling methoxy poly(ethylene glycol)-
palmitate nanocarrier for curcumin delivery to cancer cells. Acta
Biomater 4:1752–1761
46. Butterfield DA, Stadtman ER (1997) Protein oxidation processes
in aging brain. Adv Cell Aging Gerontol 2:161–191
47. Mythri RB, Jagatha B, Pradhan N et al (2007) Mitochondrial
complex I inhibition in Parkinson’s disease: how can curcumin
protect mitochondria? Antioxid Redox Signal 9:399–408
48. Tietze F (1969) Enzymic method for quantitative determination of
nanogram amounts of total and oxidized glutathione: applications
to mammalian blood and other tissues. Anal Biochem 27:502–522
49. Singh NP, McCoy MT, Tice RR et al (1988) A simple technique
for quantitation of low levels of DNA damage in individual cells.
Exp Cell Res 175:184–191
50. Trounce IA, Kim YL, Jun AS et al (1996) Assessment of mito-
chondrial oxidative phosphorylation in patient muscle biopsies,
lymphoblasts, and transmitochondrial cell lines. Methods Enzy-
mol 264:484–509
51. Pandareesh MD, Anand T (2013) Neuromodulatory propensity
of Bacopa monniera against scopolamine-induced cytotoxicity
in PC12 cells via down-regulation of AChE and up-regulation of
BDNF and muscarnic-1 receptor expression. Cell Mol Neurobiol
33:875–884
31. Liu A, Lou H, Zhao L et al (2006) Validated LC/MS/MS assay for
curcumin and tetrahydrocurcumin in rat plasma and application
to pharmacokinetic study of phospholipid complex of curcumin.
J Pharm Biomed Anal 40:720–727
32. Bishnoi M, Chopra K, Rongzhu L et al (2011) Protective effect
of curcumin and its combination with piperine (bioavailability
enhancer) against haloperidol-associated neurotoxicity: cellular
and neurochemical evidence. Neurotox Res 20:215–225
33. Suresh D, Srinivasan K (2010) Tissue distribution and elimina-
tion of capsaicin, piperine and curcumin following oral intake in
rats. Indian J Med Res 131:682–691
34. Harish G, Venkateshappa C, Mythri RB et al (2010) Bioconju-
gates of curcumin display improved protection against gluta-
thione depletion mediated oxidative stress in a dopaminergic
neuronal cell line: implications for Parkinson’s disease. Bioorg
Med Chem 18:2631–2638
35. Furniss BS (1989) Vogel’s textbook of practical organic chemis-
try. (Pearson Education India), 5th edn. Longman Ltd., Harlow,
p 654
36. Mohammed AI, Jwad RS (2011) J Kerbala Univ 9:68687
37. Joshi VY, Sawant MR (2006) A convenient stereoselective syn-
thesis of beta-d-glucopyranosides. Indian J Chem 45:461–465
38. Mohri K, Watanabe Y, Yoshida Y et al (2003) Synthesis of glyco-
sylcurcuminoids. Chem Pharm Bull (Tokyo) 51:1268–1272
39. Hergenhahn M, Bertram B, Wiessler M et al (2003) Curcumin
derivatives with improved water solubility compared to curcumin
and medicaments containing the same. US PatentApplication No.
10/312,951
40. Vali S, Mythri RB, Jagatha B et al (2007) Integrating glutathi-
one metabolism and mitochondrial dysfunction with implica-
tions for Parkinson’s disease: a dynamic model. Neuroscience
149:917–930
52. Pandareesh MD, Anand T (2014) Neuroprotective and anti-
apoptotic propensity of Bacopa monniera extract against sodium
nitroprusside induced activation of iNOS, heat shock proteins and
apoptotic markers in PC12 cells. Neurochem Res 39:800–814
53. Hosamani R, Muralidhara (2009) Neuroprotective efficacy of
Bacopa monnieri against rotenone induced oxidative stress and
neurotoxicity in Drosophila melanogaster. Neurotoxicology
30:977–985
54. Feany MB, Bender WW (2000) A Drosophila model of Parkin-
son’s disease. Nature 404:394–398
55. Linhart R, Wong SA, Cao J et al (2014) Vacuolar protein sorting
35 (Vps35) rescues locomotor deficits and shortened lifespan in
Drosophila expressing a Parkinson’s disease mutant of Leucine-
Rich Repeat Kinase 2 (LRRK2). Mol Neurodegener 9:23
56. Luo D, Zhang Q, Wang H et al (2009) Fucoidan protects against
dopaminergic neuron death in vivo and in vitro. Eur J Pharmacol
617:33–40
57. Bradford MM (1976) A rapid and sensitive method for the quan-
titation of microgram quantities of protein utilizing the principle
of protein–dye binding. Anal Biochem 72:248–254
58. Bollimpelli VS, Kumar P, Kumari S et al (2016) Neuroprotective
effect of curcumin-loaded lactoferrin nano particles against rote-
none induced neurotoxicity. Neurochem Int 95:37–45
59. Madathil KS, Karuppagounder SS, Haobam R et al (2013) Nitric
oxide synthase inhibitors protect against rotenone-induced,
oxidative stress mediated parkinsonism in rats. Neurochem Int
62:674–683
60. Dinkova-Kostova AT, Talalay P (2010) NAD(P)H:quinone accep-
tor oxidoreductase 1 (NQO1), a multifunctional antioxidant
enzyme and exceptionally versatile cytoprotector. Arch Biochem
Biophys 501:116–123
61. Ross D, Siegel D (2004) NAD(P)H:quinone oxidoreductase 1
(NQO1, DT-diaphorase), functions and pharmacogenetics. Meth-
ods Enzymol 382:115–144
41. Wroblewski F, Ladue JS (1955) Lactic dehydrogenase activity in
blood. Proc Soc Exp Biol Med 90:210–213
1 3

16
Neurochem Res
62. Martinez TN, Greenamyre JT (2012) Toxin models of mitochon-
drial dysfunction in Parkinson’s disease. Antioxid Redox Signal
16:920–934
76. Yu S, Zheng W, Xin N et al (2010) Curcumin prevents dopami-
nergic neuronal death through inhibition of the c-Jun N-terminal
kinase pathway. Rejuvenation Res 13:55–64
63. Vassallo N (2008) Polyphenols and health: new and recent
advances. Nova Publishers, New York
64. Prasad S, Tyagi AK, Aggarwal BB (2014) Recent developments
in delivery, bioavailability, absorption and metabolism of cur-
cumin: the golden pigment from golden spice. Cancer Res Treat
46:2–18
65. Sharma RA, Steward WP, Gescher AJ (2007) Pharmacokinet-
ics and pharmacodynamics of curcumin. In: The molecular tar-
gets and therapeutic uses of curcumin in health and disease, pp
453–470
66. Gadad BS, Subramanya PK, Pullabhatla S et al (2012) Curcumin-
glucoside, a novel synthetic derivative of curcumin, inhibits
alpha-synuclein oligomer formation: relevance to Parkinson’s
disease. Curr Pharm Des 18:76–84
77. ChenYR, Tan TH (1998) Inhibition of the c-Jun N-terminal kinase
(JNK) signaling pathway by curcumin. Oncogene 17:173–178
78. Schaeffer HJ, Weber MJ (1999) Mitogen-activated protein
kinases: specific messages from ubiquitous messengers. Mol Cell
Biol 19:2435–2444
79. Dinkova-Kostova AT, Talalay P (2000) Persuasive evidence that
quinone reductase type 1 (DT diaphorase) protects cells against
the toxicity of electrophiles and reactive forms of oxygen. Free
Radical Biol Med 29:231–240
80. Radjendirane V, Joseph P, Lee YH et al (1998) Disruption of the
DT diaphorase (NQO1) gene in mice leads to increased menadi-
one toxicity. J Biol Chem 273:7382–7389
81. Asher G, Tsvetkov P, Kahana C et al (2005) A mechanism of
ubiquitin-independent proteasomal degradation of the tumor sup-
pressors p53 and p73. Genes dev 19:316–321
67. Mishra S, Narain U, Mishra R et al (2005) Design, development
and synthesis of mixed bioconjugates of piperic acid-glycine,
curcumin-glycine/alanine and curcumin-glycine-piperic acid and
their antibacterial and antifungal properties. Bioorg Med Chem
13:1477–1486
82. Tsvetkov P, Asher G, Reiss V et al (2005) Inhibition of NAD(P)
H:quinone oxidoreductase 1 activity and induction of p53 deg-
radation by the natural phenolic compound curcumin. Proc Natl
Acad Sci USA 102:5535–5540
68. Liang C, Zhang Y, Jia Y et al (2016) Engineering a carbohydrate-
processing transglycosidase into glycosyltransferase for natural
product glycodiversification. Sci Rep 6:21051
83. Gaikwad A, Long DJ 2nd, Stringer JL et al (2001) In vivo role of
NAD(P)H:quinone oxidoreductase 1 (NQO1) in the regulation of
intracellular redox state and accumulation of abdominal adipose
tissue. J Biol Chem 276:22559–22564
69. Nataraj J, Manivasagam T, Justin Thenmozhi A et al (2016)
Neuroprotective effect of asiatic acid on rotenone-induced mito-
chondrial dysfunction and oxidative stress-mediated apoptosis in
differentiated SH-SYS5Y cells. Nutr Neurosci 9:1–9
70. Sala G, Marinig D, Riva C et al (2016) Rotenone down-regulates
HSPA8/hsc70 chaperone protein in vitro: A new possible toxic
mechanism contributing to Parkinson’s disease. Neurotoxicology
54:161–169
71. Zhang Q, Chen S, Yu S et al (2016) Neuroprotective effects of
pyrroloquinoline quinone against rotenone injury in primary cul-
tured midbrain neurons and in a rat model of Parkinson’s disease.
Neuropharmacology 108:238–251
84. Tsvetkov P, Adamovich Y, Elliott E et al (2011) E3 ligase STUB1/
CHIP regulates NAD(P)H:quinone oxidoreductase 1 (NQO1)
accumulation in aged brain, a process impaired in certain
Alzheimer disease patients. J Biol Chem 286:8839–8845
85. Lee SC, Zhao ML, Hirano A et al (1999) Inducible nitric oxide
synthase immunoreactivity in the Alzheimer disease hippocam-
pus: association with Hirano bodies, neurofibrillary tangles, and
senile plaques. J Neuropathol Exp Neurol 58:1163–1169
86. Luth HJ, Munch G, Arendt T (2002) Aberrant expression of NOS
isoforms in Alzheimer’s disease is structurally related to nitroty-
rosine formation. Brain Res 953:135–143
72. Turrens JF (2003) Mitochondrial formation of reactive oxygen
species. J Physiol 552:335–344
87. Iadecola C, Alexander M (2001) Cerebral ischemia and inflam-
mation. Curr Opin Neurol 14:89–94
73. Jagatha B, Mythri RB, Vali S et al (2008) Curcumin treatment
alleviates the effects of glutathione depletion in vitro and in vivo:
therapeutic implications for Parkinson’s disease explained via in
silico studies. Free Radical Biol Med 44:907–917
74. Li N, Ragheb K, Lawler G et al (2003) Mitochondrial complex
I inhibitor rotenone induces apoptosis through enhancing mito-
chondrial reactive oxygen species production. J Biol Chem
278:8516–8525
75. Kelso GF, Porteous CM, Coulter CV et al (2001) Selective tar-
geting of a redox-active ubiquinone to mitochondria within
cells: antioxidant and antiapoptotic properties. J Biol Chem
276:4588–4596
88. Park EM, Cho S, Frys K et al (2004) Interaction between induc-
ible nitric oxide synthase and poly(ADP-ribose) polymerase in
focal ischemic brain injury. Stroke 35:2896–2901
89. Coulom H, Birman S (2004) Chronic exposure to rotenone mod-
els sporadic Parkinson’s disease in Drosophila melanogaster. J
Neurosci 24:10993–10998
90. Beal MF (2003) Bioenergetic approaches for neuroprotection in
Parkinson’s disease. Ann Neurol 53(Suppl 3):S39–47 (discussion
S38–47)
91. Celotto AM, Palladino MJ (2005) Drosophila: a “model” model
system to study neurodegeneration. Mol Interv 5:292–303
1 3