Accepted Manuscript

Neurorestorative effects of eugenol, a spice bioactive: Evidence in cell model and its
efficacy as an intervention molecule to abrogate brain oxidative dysfunctions in the
streptozotocin diabetic rat

N. Sathya Prasad, M.M.S. Bharath, Muralidhara

PII: S0197-0186(15)30062-0
DOI: 10.1016/j.neuint.2015.10.012
Reference: NCI 3783

To appear in: Neurochemistry International

Received Date: 18 April 2015

Revised Date: 9 October 2015
Accepted Date: 24 October 2015

Please cite this article as: Sathya Prasad, N, Bharath, M., Muralidhara, Neurorestorative effects of
eugenol, a spice bioactive: Evidence in cell model and its efficacy as an intervention molecule to
abrogate brain oxidative dysfunctions in the streptozotocin diabetic rat, Neurochemistry International
(2015), doi: 10.1016/j.neuint.2015.10.012.

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Revised Version
Neurorestorative effects of Eugenol, a spice bioactive: evidence in cell model and its
efficacy as an intervention molecule to abrogate brain oxidative dysfunctions in the
streptozotocin diabetic rat

Sathya Prasad N1, Bharath MMS2 and Muralidhara1

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1
Dept of Biochemistry & Nutrition, CSIR - Central Food Technological Research Institute
(CFTRI), Mysuru- 570020, India

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2
Department of Neurochemistry,

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National Institute of Mental Health and Neurosciences (NIMHANS),
#2900, Hosur Road, Bengaluru -560029, India

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Address for correspondence:

Dr. Muralidhara
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Chief Scientist
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Department of Biochemistry and Nutrition,

CSIR - Central Food Technological Research Institute,
Mysore - 570020, Karnataka, India
TEL: 91-821-2514876; FAX: 91-821-2517233
Email: mura16@yahoo.com
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ABSTRACT

Eugenol (EU), an active principle of cloves, is also widely distributed in various other
plants (eg. basil, cinnamon etc). While its antioxidant and anti-inflammatory properties are
well established, biochemical insights related to its neuromodulatory potential in diabetic
conditions are not clear. In the present study, initially we investigated its potential to

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modulate specific biochemical responses in SH-SY5Y cells under experimentally -induced
hyperglycemic condition. Co-exposure of cells with EU (5 - 10 µM) not only enhanced the

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cell viability, but significantly offset glucose -associated oxidative stress (as evidenced by
diminished levels of reactive oxygen species and hydroperoxides). Further EU enhanced the

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reduced glutathione (GSH) levels and also ameliorated the levels of 3 – nitrotyrosine and
expression of HSP70. We subsequently examined its efficacy to attenuate biochemical
aberrations in brain regions of a streptozotocin (STZ) diabetic rat employing an intervention

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approach. Brain regions of EU treated (10 mg/ kg bw/d, post 6 weeks of STZ) diabetic rats
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showed diminished levels of oxidative markers and protein carbonyls in both cytosolic and
mitochondrial fractions. EU treatment caused enhanced activities of enzymic antioxidants and
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diminished both GSH and total thiols. Further, activities of complex I – III, succinate
dehydrogenase and citrate synthase in brain regions were also significantly restored.
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Interestingly, EU treatment differentially attenuated the elevated activity of

acetylcholinesterase and levels of calcium in brain regions. Collectively, based on the data
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obtained in in vitro and in vivo models, we hypothesize that EU may be employed as an

adjuvant therapeutic molecule to alleviate complications under diabetic conditions.
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Key words:

Eugenol, SHSY5Y cells, rat, diabetes, brain oxidative impairments, mitochondrial

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dysfunctions
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1. Introduction

Diabetes, a chronic metabolic disorder, is a leading cause of morbidity, mortality and

disability. The International Diabetic Federation (IDF) estimates that 438 million people
world-wide would suffer from diabetes by the year 2030 (International Diabetes Federation,
2011). One of the most common complications of diabetes mellitus is diabetic neuropathy
(DN), a microvascular complication leading to the damage of the nervous system and 50-60%

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diabetics exhibit some form of neurological disorder (Obrosova, 2009). The neurochemical,
neurophysiological and structural aberrations resulting from hyperglycemic effects in the

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central nervous system (CNS) is referred to as diabetic encephalopathy (DE) (Kamboj and
Sandhir, 2011; Urban et al., 2012). Alterations in the mitochondrial and endoplasmic

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reticulum have been associated with cytosolic aberrations leading to events such as energy
depletion in the development of DN complications (Chowdhury et al, 2013; Cameron, 2013).
Common symptoms among diabetics include numbness, tingling, allodynia and hyperalgesia.

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DN, to-date has no definite pharmacological solution due to its multifactorial etiology (Yorek,
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2011).

Several experimental models have been employed to obtain insights related to the
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implications of hyperglycemic effects and for developing therapeutic strategies (Hattangady

and Rajadhyaksha, 2009). One of the cell lines often used for the purpose is SHSY5Y cells, a
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cloned sub-line of the neuroblastoma SK-N-SH cell line established from metastatic bone
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tumour. Excessive free radical production and the related mitogen-activated protein kinase
P38 activation have been suggested as key regulators in the pathogenesis of high glucose-
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induced cell stress in SHSY5Y cells (Cao et al., 2012). It is speculated that the fluctuating
glucose levels in SHSY5Y cells had a greater adverse effect on energy turn-over than either
persistent high or persistent low glucose levels (Russo et al., 2012). Chronic hyperglycemia
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generates reactive oxygen species (ROS) in part through the formation and downstream
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signalling effects of advanced glycation end products (AGEs). In cultured cells, AGEs are
shown to promote oxidative damage and apoptosis (Pazdro and Burgess, 2012).

Accumulating evidence (both experimental and clinical) suggest that polyphenols-rich

natural products, like nutraceuticals and food supplements, may offer unique treatment
modalities in type 2 diabetes mellitus. A combinatorial therapy that targets causal
mechanism/s and enhances endogenous reparative capacity may alleviate nerve function and
regeneration under diabetic condition. Further, phyto-constituents potentially decrease
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oxidative stress and possibly improve mitochondrial bioenergetics (Urban et al., 2012). Hence
search for new therapeutic interventions has been the subject of intense research among
experimenters world-wide. Various herbal actives and spice bioactives have been in use either
per se or in various formulations in different forms of traditional and complementary
medicines such as Ayurveda. Phyto-constituents such as curcumin, cinnamaldehyde,
berberine, withanoloides, bacopasides have exhibited preventive or curative properties against

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DN (Parihar et al., 2004; Sharma et al., 2006; Cao et al., 2010). These are presumably less
toxic and well tolerated than oral hypoglycemic agents which have been demonstrated to

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cause various side effects in humans.

Eugenol (EU, 4-allyl-2-methoxyphenol), an active principle of cloves, is found in various

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other plants such as basil, cinnamon and other herbs. It is widely used as flavouring agent in
baked products, beverages, sweets and frozen dairy products. In ayurvedic practices it is used

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as an antiseptic, carminative and analgesic for dental problems. EU is a well-known
antioxidant and anti-inflammatory agent whose potential to abrogate oxidative stress and
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inflammatory reactions is well established (Mahapatra et al., 2009; Hidalgo and Rosa, 2009;
Baskaran et al., 2010; Prasad and Muralidhara, 2013). Previously EU was shown to possess
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inhibitory effects on excitotoxin induced oxidative damage, neurotoxicity and convulsions

(Wie et al., 2006). The depressant activity of EU on CNS and the local anesthetic effect have
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also been well documented (Guenette et al., 2006; Reiner et al., 2013). While there have been
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few reports on the protective effects of EU in diabetic models, limited data exists on its
neurorestorative efficacy (Mnafgui et al, 2013; Sanae et al, 2014). To the best of our
knowledge, the efficacy of EU to attenuate oxidative impairments and mitochondrial
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dysfunctions in brain regions following induction of diabetes employing a intervention

paradigm has not been previously addressed.
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In view of this, initially we determined the potential of EU to offset hyperglycemia

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associated oxidative stress and cell viability in SHSY5Y cells. Subsequently, we sought to
assess the neuro-ameliorative effects of EU in STZ-diabetic model employing an intervention
approach. In this model, we validated its potential to attenuate oxidative stress, redox status,
protein oxidation, mitochondrial dysfunctions and cholinergic functions in brain regions.
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2. Materials and Methods

2.1. Chemicals
Streptozotocin, eugenol, thiobarbituric acid (TBA), 1,1,3,3- Tetra methoxypropane,
2’,7’-dichloro-fluorescein (DCF), 2’,7’-dichloro-fluorescein diacetate and other fine
chemicals were procured from M/s Sigma Chemical Co. (St Louis, MO, USA). All other
chemicals used were of analytical grade.

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2.2. Protective effect of Eugenol (EU) in SHSY5Y cells

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2.2.1. Induction of hyperglycemia

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SHSY5Y, a human neuroblastoma cell line, was maintained in DMEM media
supplemented with 10% FBS and penstrep. The growth conditions include humid atmosphere

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of 5% CO2 and 95% O2 at 37oC. Cells were plated in 96 well plates at a density of 5 x 105
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cells/ well. Cells were exposed to different concentrations of glucose (Glc; 25 to 300 mM) for
24 h. Cell survival was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
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diphenyltetrazoliumbromide] assay (Berridge and Tans, 1993). The MTT assay (, reduction of
tetrazolium salts) is a measure of mitochondrial activity which quantitatively reflects cell
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viability (Russo et al., 2012). The yellow tetrazolium MTT gets reduced by metabolically
active cells, in part by the action of dehydrogenase enzymes resulting in the formation of
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purple formazan crystals. These intracellular formazan crystals were then solubilized in SDS-
DMF buffer (45% DMF in distilled water and 10% SDS, pH 4.7). It was quantified
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spectrophotometrically at 570 nm and per cent cell survivability was calculated against
control (using OD as a measure).
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2.2.2. Effect of EU on cell survivability

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A 10 mM stock solution of EU was prepared in 95% ethanol, while all the working
standards were prepared in the culture media. Initially cells were plated in 96 well plates at a
density of 5 x 105 cells/ well containing different concentrations (1 to 100 µM) of EU for 24
h. Cell survival was determined by MTT assay in order to ascertain the non-toxic
concentrations. These concentrations were employed to assess the potential to modulate
glucose (Glc) induced cellular aberrations and cell death.
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2.2.3. Modulatory effect of EU

Following co-exposure of cells to sub-lethal concentrations of EU and 100 mM of Glc

(IC50 concentration) for 24 h cell survival and protection rendered by the actives against Glc
induced cell death was determined by MTT assay. For biochemical estimations, experiments
were carried out only with selected concentrations of EU (those which they rendered

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protection against Glc induced cell death). Selected markers for oxidative stress (gneenration
of ROS, levels of hydroperoxides (HP) and GSH) were determined post 24 h co-exposure

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among various treatment groups. Further, the modulatory effect of EU on the levels of 3-
nitrotyrosine (3-NT) and HSP70 were determined using slot blot analysis.

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2.2.4. Measurement of Nitrated Proteins (3-NT)

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The extent of protein nitration was estimated in terms of 3-nitrotyrosine (3NT) employing
a slot-blot method. In brief, aliquots (15 µg protein) were spotted in triplicates onto a
nitrocellulose membrane. The membranes were treated with a blocking buffer containing 2 %
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BSA in PBST (PBS, 0.1 M, pH 7.4, containing 0.14 M NaCl and 0.01 % Tween-20) for 60
min at room temperature and probed with polyclonal anti-3-NT antibody (1:500; origin-
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Rabbit; Sigma Chemical Co. USA.) over the next 60 min. The membranes were washed
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repeatedly in PBST and incubated with ALP-conjugated secondary antibody (1:3000; origin-
goat; Merck-Bangalore Genei, India) for the last 60 min. The membranes were developed
employing BCIP/ NBT reaction. To adjust for protein loading, duplicate membranes were
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also immune-stained with anti- β actin monoclonal antibody (1:2000; Sigma, USA). Optical
density on the blots was measured with Image J Software (NIH, USA). The extent of protein
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nitration was expressed as arbitrary units (Chandran and Muralidhara, 2013).

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2.2.5. Measurement of HSP70

The expression of HSP70 was estimated using slot-blot analysis. In brief, aliquots (20 µg
protein) were spotted in triplicates onto a nitrocellulose membrane using a slot-blot apparatus.
The membranes were treated with a blocking buffer containing 2 % BSA in PBST (PBS, 0.1
M, pH 7.4, containing 0.14 M NaCl and 0.01 % Tween-20) for 60 min at room temperature.
After washing, the blots were probed with monoclonal anti-HSP70 antibody (1:500; origin-
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mouse) for 3 h. The membranes were washed repeatedly in PBST and incubated with ALP-
conjugated secondary antibody (1:3000; anti-mouse) for 60 min. The membranes were
developed employing BCIP/ NBT reaction. To adjust for protein loading, duplicate
membranes were also immune-stained with anti- β actin monoclonal antibody (1:2000;
Sigma, USA). Optical density on the blots was measured with ImageJ Software (NIH, USA).
The expression of HSP70 was presented as arbitrary units (Mythri et al., 2011).

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2.3. Neurorestorative effect of Eugenol in STZ Diabetic rat model

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2.3.1. Animal care

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Adult (8-9 wks old) male albino rats (CFT-Wistar strain) were drawn from the stock
colony of Institute Animal Facility. Animals were maintained on commercial chow diet and
water ad libitum. All experiments were conducted strictly in accordance with approved

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guidelines by the ‘Institute Animal Ethical Committee’ regulated by the Committee for the
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Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of
India, India (Registration number: 49/1999/CPCSEA).
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2.3.2. Experimental design

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Diabetes was induced by a single injection of freshly dissolved STZ (55 mg/ kg of body
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weight) in a 0.1 mol/ L citrate buffer (pH 4.5) into the peritoneum. Control rats were injected
with citrate buffer. STZ injected rats were provided with 5% glucose in drinking water for 48
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h. Three days after STZ injection, rats were screened for blood glucose levels employing
Accuchek comfort sensor glucometer. Animals with glucose levels ≥ 350 mg/ dL after 72 h of
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STZ injection were included in the study.

After six weeks of post STZ, rats were grouped as follows: Control rats were divided into
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two groups: Group I – Control; Group II – EU control (EU, 10 mg/ kg bw/ every alternate
day, ip., 6 weeks). Diabetic rats were divided into two groups: Group III – Diabetic untreated;
Group IV – Diabetic rats administered with EU (Diabetic + EU, 10 mg/ kg bw/ alternate days,
ip., 6 weeks). Group I and Group III rats received equi-volume of DMSO. The dosage of EU
was based on our previous findings (Prasad and Muralidhara, 2013). Daily feed intake and
weekly body weights were recorded throughout the experimental period of 12 weeks. Rats of
all the groups were tested for blood glucose levels using glucometer bi-weekly. Terminally,
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rats were sacrificed under mild anaesthesia; brain was excised and processed on ice for
biochemical analysis.

2.4. Biochemical analysis in brain regions

Brain regions – cortex (Ct), cerebellum (Cb), striatum (St) and hippocampus (Hc) were

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dissected over ice. A 10% homogenate of brain regions were prepared in ice-cold tris-Sucrose
buffer (0.25 M, pH 7.4) and centrifuged at 1000 x g for 10 min at 4°C to obtain the nuclear
pellet. Differential centrifugation (700 and 4500 x g, 10 min, 4o C) was employed to isolate

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cytosol and mitochondria from different brain regions (Moreadith and Fiskum, 1984). The

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crude mitochondrial pellet was washed and re-suspended using HEPES buffer (HEPES - 10
mM, EDTA - 0.1 mM, mannitol - 200 mM, sucrose - 70 mM; pH 7.4). All samples were
stored in aliquots at -80oC till next use.

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2.4.1. Measurement of ROS generation
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ROS generation was assayed using 2’,7’-dichloro-fluorescein diacetate (H2 DCFH-DA), a

non-polar compound, which after conversion to a polar derivative by intracellular esterases,
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rapidly reacts with ROS to form the highly fluorescent compound dichlorofluorescein (DCF)
(Chandrashekar and Muralidhara, 2008). Briefly, an aliquot (100 µg protein equivalent) was
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incubated in Locke’s buffer (pH 7.4; NaCl - 154 mM, KCl - 5.6 mM, NaHCO3 - 3.6 mM,
HEPES - 5 mM, CaCl2 - 2 mM and glucose - 10 mM) containing H2DCFH-DA (5 µM) for 30
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min at room temperature. The fluorescent product DCF formed was measured using a
spectrofluorimeter with an excitation and emission wave lengths of 480 nm and 530 nm
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respectively. The ROS generation was calculated from a DCF standard curve and expressed
as ρmol DCF/ min/ mg protein.
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2.4.2. Measurement of hydroperoxides (HP)

HP levels were measured according to a previously described method using FOX 1

reagent with minor modifications (Wolff, 1994). An aliquot of cytosolic (or mitochondrial)
fraction (100 µg protein) was added to 1 mL FOX reagent (100 µM xylenol orange; 250 µM
ammonium ferrous sulphate; 100 µM sorbitol; 25 mM H2SO4) and incubated for 30 min at
room temperature. The colour developed was read at 560 nm in a spectrophotometer. The
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concentration of HP was calculated using the molar extinction co-efficient (ε = 2.2 × 105 M-1
cm-1) and expressed as nmol HP/ mg protein.

2.4.3. Assessment of lipid peroxidation (LPO)

LPO was assessed by measuring the formation of thiobarbituric acid reactive substances

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(TBARS) following the method described previously (Ohkawa et al., 1979). Briefly, the
reaction mixture contained an aliquot of cytosolic/ mitochondrial fraction of different brain

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regions (500 µg protein), 1.5 mL of acetic acid (pH 3.5, 20 %), 1.5 mL of 0.8 % thiobarbituric
acid (0.8 % w/v) and 0.2 mL SDS (8% w/v). The mixture was heated to boiling for 45 min

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and TBARS adducts were measured at 532 nm and quantified as malondialdehyde (MDA)
equivalents using 1,1,3,3-tetramethoxypropane as the standard.

2.4.4. Measurement of nitric oxide (NO)

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NO was estimated using as Griess reagent (M/s Sigma Chemicals) as per specifications in
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the catalogue. Briefly, to an aliquot of cytosol/ mitochondrial fraction (100 µg protein) Griess
reagent was added, the coloured product formed was read at 540 nm. NO was quantified as
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nitrites using a standard curve.

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2.4.5. Determination of protein carbonyls (PC)

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PC levels in the samples were quantified by the method of Levine et al., (1990). Briefly
an aliquot (500µg protein) of cytosolic (or mitochondrial) fraction was incubated with 2, 4-
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dinitrophenyl hydrazine for 1h. The protein was precipitated by adding trichloroacetic acid
(20%) followed by centrifugation. The pellet washed twice with acetone and dissolved in 2
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mM Tris-HCl buffer (pH 7.4; containing 200 mM NaCl and 2% SDS). The optical density
was measured at 370 nm and expressed as nmol carbonyl/ mg protein (ε = 22000 M-1cm-1).

2.4.6. Estimation of reduced glutathione (GSH)

GSH content was quantified based on a fluorimetric method described previously

(Mokrasch and Teschke 1984) using o-phthalaldehyde (OPT). Briefly an aliquot of cytosolic
fraction was added to formic acid (0.1 M) and centrifuged at 10,000 × g for 10 min. An
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aliquot of supernatant (de-proteinized) was added to tubes containing buffered formaldehyde
(1: 4 (v/v) 37% formalin: 0.1 M Na2HPO4). 0.1 M Sodium phosphate buffer (pH 8.0;
containing 5 mM EDTA) was added to each tube followed by OPT (100 µg/ mL). Following
incubation for 45 min at room temperature, the fluorescence was measured at excitation and
emission wavelengths of 345 and 425 nm respectively. Concentration of GSH was calculated
from a standard curve and values were expressed as µg /mg protein.

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2.4.7. Estimation of total thiols (TSH)

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The ability of thiols to oxidize 5,5-dithiobis 2- nitrobenzoic acid (DTNB) was measured

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spectrophotometrically at 412 nm (Ellman, 1959). An aliquot (50 µg) of the sample (cytosol/
mitochondria) was added to Tris buffer (0.2M, pH 8.2) containing 25 µL of DTNB (10 mM in
methanol) and 1.975 mL of methanol. Following incubation for 30 min at RT, the tubes were

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centrifuged at 3000 x g for 10 min. The clear supernatant was read at 412 nm against distilled
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water and expressed as nmol substrate oxidized/ mg protein (ε = 13.6 mM-1cm-1).
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2.4.8. Activities of selected enzymes

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Catalase (CAT) activity was determined according to a previously described method

(Aebi, 1984). The reaction was initiated by adding an aliquot of the cytosol (equivalent to 50
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µg protein) to 1 mL reaction mixture containing 8.8 mM H2O2 (3%), 0.1 mM sodium

phosphate buffer (pH 7). The decrease in H2O2 was monitored for 3 min at 240 nm and
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expressed as nmol of H2O2 decomposed/ min/ mg protein (ε = 43.6 mM-1cm-1).

Superoxide dismutase (SOD) activity was determined by monitoring the inhibition of
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quercetin auto-oxidation. The reaction was started by adding an aliquot of quercetin (stock:1.5
mg in 1 mL dimethyl formamide) to a volume of 1 mL reaction mixture containing 3–5 µg
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protein; 0.016M sodium phosphate buffer (pH 7.8), 8 mM N,N,N,N tetra methyl
ethylenediamine (TEMED) and 0.08 mM EDTA. Reaction was monitored for 3 min at 406
nm. The activity (U) has been expressed as the amount of protein required to inhibit 50% of
quercetin auto-oxidation (Kostyuk and Potapovich, 1989).
Glutathione–S–Transferase (GST) activity was quantified (Guthenberg et al., 1985) by
monitoring the conjugation of GSH to 1-Chloro-2,4-dinitrobenzene (CDNB) at 340 nm. The
reaction was initiated by adding a cytosolic aliquot (0.01 mg protein) to 0.1M phosphate
buffer (pH 6.5; containing in 0.5 mM EDTA, 0.075 mM CDNB, 0.05 mM GSH). The
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increase in the optical density at 340 nm was recorded for 3 min and the activity expressed as
nmol conjugate formed/ min/mg protein (ε = 9.6m M-1cm-1).
Glutathione reductase (GR) activity was measured (Carlberg et al., 1984) by the addition
of cytosolic aliquot (0.2 mg protein) to a reaction mixture of 0.2 M phosphate buffer (pH 7.0)
containing 2 mM EDTA, 20 mM oxidized glutathione and 2 mM NADPH. The decrease in
the absorbance at 340 nm due to oxidation of NADPH was monitored for 3 min and the

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activity expressed as nmol NADPH oxidized/ min/ mg protein (ε = 6.22 mM-1cm-1).
Thioredoxin reductase (TRR) activity (Luthman and Holmgen, 1982) in the test sample

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was measured by monitoring the reduction of DTNB at 412 nm, in a potassium phosphate
buffer (0.1M, pH 7.0, containing 10 mM EDTA, 0.2 mM NADPH). The activity was
expressed as nmol substrate reduced/ min/ mg protein (ε = 13.6m M-1cm-1).

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2.4.9. Cytosolic calcium levels

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Intracellular calcium levels were measured in the cytosolic fraction of different brain
regions using Fura-2AM (Aoshima et al., 1997). The cytosol equivalent to 50 µg protein was
incubated in 0.1 M Tris HCl buffer (pH 7.8) for 30 min at 37oC. The fluorescence was
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measured at excitation and emission wave lengths of 488 nm and 525 nm respectively. The
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amount of calcium was calculated using a standard curve and expressed as ng/ mg protein.
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2.4.10. Assays in mitochondrial fraction

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MTT reduction was carried out as per the method of Berridge and Tans (1993). Briefly
mitochondrial sample (equivalent to 10 µg protein) was added to 1000 µL of buffer
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(Mannitol-Sucrose-HEPES, 20mM sodium succinate, 1mM NADH, pH 7.4). To this 15 µl of

MTT (5 mg/ mL) was added and incubated at 37oC for 1 h. The formazan crystals formed
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were dissolved in SDS-DMF buffer (45% DMF in distilled water and 10% SDS, pH 4.7), and
the absorbance was read at 570 nm.
To assess the activity of NADH - Cyt C reductase (Complex I - III), an aliquot of
mitochondria (50 µg) was added to phosphate buffer (0.1 M, pH 7.4) containing NADH - 0.2
mM and KCN - 1 mM. The reaction was initiated by addition of 0.1 mM cytochrome C and
the decrease in absorbance was monitored for 3 min at 550 nm. The activity was expressed as
nmol cytochrome C reduced/ min/mg protein (ε = 19.6 mM-1cm-1) (Navarro et al., 2004).
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Succinate dehydrogenase (SDH) activity was determined as described earlier
(Pennington, 1961) with minor modifications. Briefly, mitochondrial protein (50 µg) was
incubated with 50 mM potassium phosphate buffer (pH 7.4) containing sodium succinate
(0.01mol/ L) and p‐iodonitrotetrazolium violet (2.5 µg/ mL) for 10 min. The reaction was
stopped by addition of 10% TCA. The colour obtained was extracted with ethyl acetate:
ethanol: trichloroacetic acid (5:5:1, v:v:v) and measured at 490 nm. The activity was

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expressed as OD/ mg protein.
Citrate synthase (CS) activity was determined by the oxidation of DTNB as described by

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Srere, 1969. Mitochondrial protein (50 µg) was added to Tris–HCl buffer (0.1 M, pH 8.1,
0.1% Triton X-100) containing DTNB (0.2 mM) and acetyl CoA (0.1 mM). The reaction was

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started by the addition of oxaloacetate (10 mM) and absorbance was monitored at 412 nm for
3 min. The activity was expressed as nmol substrate conjugated/ min/ mg protein (ε = 13.6
mM-1cm-1).

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2.4.11. Activity of acetylcholinesterase (AChE)
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The activity of AChE was determined (Ellman et al. 1961) by taking a reaction mixture
containing 0.1 M phosphate buffer (pH 8.0), 10 mM DTNB, an aliquot of cytosol (100 µg
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protein) and acetylthiocholine iodide (150 mM). The change in absorbance was monitored at
412 nm for 3 min. The enzyme activity was expressed as nmol of substrate hydrolyzed/ min/
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mg protein (ε = 13.6mM-1cm-1).
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2.5. Determination of protein

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Protein concentrations in all test samples were determined by incubating an aliquot of the
sample with Folin – Ciocalteau’s phenol in an alkaline medium (30 min) and measuring the
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OD at 750 nm using a UV-Visible spectrophotometer (Lowry et al., 1951). The amount of

protein was quantified using bovine serum albumin as the standard.

2.6. Statistical analysis

Results are represented as the group means ± standard error (SE) for each experimental
group. The data was analysed by one-way ANOVA followed by a post hoc ‘Tukey’ test to
compare the control and treatment groups. p ≤ 0.001 and/ or p ≤ 0.05 was considered
statistically significant for in vitro studies and p ≤ 0.05 was considered as statistically
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significant for in vivo study. All statistical analysis was performed using SPSS statistical
software package version 17.0.

3. Results

3.1. Cell culture experiments: In vitro model of hyperglycemia in SHSY5Y cells

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3.1.1. Effect of glucose on cell survivability and its modulation by EU co-treatment

Exposure of SHSY5Y cells to graded concentrations of Glc resulted in a concentration

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dependent cell death as evidenced by MTT reduction assay. While at 50 mM concentration
23% cell death was evident, 77% cell death ensued at 300 mM. However, non-linear

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regression analysis revealed IC50 value as 100.8 mM. Hence for studying the modulatory
potency of EU, Glc concentration of 100 mM was used (Fig. 1A).

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Exposure of cells to different concentrations of EU per se did not cause cell death in the
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range of 5 - 50 µM. Hence the concentrations used for studying the modulatory potency of
EU against hyperglycemia were in the range of 5 - 50 µM. EU in the concentration range of 5
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- 20 µM modulated Glc induced cell death (Fig. 1B) and consistent protection (22-26%) was
evident only at 5 and 10 µM concentrations (p ≤ 0.001)
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3.1.2. Modulatory effects of EU on Glc induced oxidative stress

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Glc exposure caused significant induction of oxidative stress as evidenced by elevation in

ROS (20%) and HP (50%) levels as well as depletion in GSH levels (35%). With EU
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exposure, the endogenous levels of ROS and HP were markedly diminished (Fig 2A and B).
When co-exposed with Glc, the ROS levels were reduced by 35% at both the concentrations.
On the other hand, HP levels displayed robust reduction with EU (5 µM: 62%; 10 µM: 76%).
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Although no significant alterations in the levels of GSH was apparent among EU treated cells,
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the depleted levels of GSH were significantly altered with EU in the co-exposure paradigm
(Fig. 2C). Interestingly EU treatment caused robust enhancement in the GSH levels (5 µM:
2.39 fold; 10 µM: 1.87 fold).

3.1.3. Modulatory effects of EU on 3-NT and HSP70 levels:

The endogenous levels of 3-NT were reduced by the EU treatment (Fig. 3A and B). While
Glc exposure resulted in an increase (46%) in 3-NT levels, a marked diminution (74%)
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occurred with EU at 10µM concentration. Further a marked elevation in the HSP70
expression was observed among cells exposed to Glc (91%) and EU (45%). Interestingly,
when co-exposed, Glc induced elevation of HSP70 expression levels were markedly
diminished (60%) by EU.

3.2. Modulatory effects of Eugenol in Diabetic rat model

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3.2.1. Effect of EU on body weight and blood glucose levels

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A general decrease in body weight gain was evident among both EU treated and untreated
diabetic rats. However, EU supplementation to non-diabetic rats did not affect the feed intake

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or body weight gain in comparison to control rats throughout the experimental period.
Terminally, the mean body weight of diabetic rats was ~50% lower than the controls (Body
weights (g): Control = 354 ± 3; EU = 350 ± 3; Diabetic = 173 ± 6; Diabetic + EU = 207 ± 19).

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Interestingly a significant reduction in the mean blood glucose level (mg/ dL) among EU
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treated diabetic rats was evident at the end of the experimental period (Table 1).
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3.2.2. Modulatory effect of EU on markers of oxidative stress

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With EU treatment the endogenous levels of HP and NO were significantly reduced only
in St region (Fig. 4A, B, C) and the ROS levels were diminished only in St and Hc. The
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elevated levels of HP among all the regions of brain of untreated diabetic rats were
normalized with EU treatment. While, an increase (30-50%) in the NO and ROS levels was
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observed in brain regions (except Cb region) of untreated diabetic rats, marked reduction was
evident with EU treatment. A moderate (Cb and St) to robust (Ct and Hc) increase in the
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MDA levels among the diabetic rats was observed (Fig. 5A). However among the EU treated
diabetic rats a marked reduction was evident in Ct and Hc, while normalizing the same in Cb
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and St (Fig. 5A). The protein oxidation levels (PC) elevated significantly among untreated
diabetic rats was apparently normalized with EU treatment (except in Hc) (Fig. 5B).

3.2.3. EU modulates cytosolic levels of calcium

A robust elevation (2-3 folds) in the cytosolic calcium levels was evident in different
regions of the brain among diabetic rats. Interestingly, EU supplementation normalized the
calcium levels in Cb and significantly ameliorated the same in other brain regions (Fig. 5C).
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3.2.4. Effect of EU on redox status

While a marked depletion (30%) in the levels of GSH occurred in all the brain regions of
diabetic rats, TSH levels were depleted only in St (Table 2). While the GSH levels were
restored in St, it was elevated further marginally in other brain regions by EU treatment. A

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marked elevation in the TSH levels was evident in Ct (38%) and Hc (56%) and a marginal
increase ensued in St of EU treated diabetic rats (Table 2).

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3.2.5. Effect of EU on the activities of antioxidant/ detoxifying enzymes

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The endogenous activity levels of some of the important redox enzymes were altered only
in few regions of the brain with EU treatment (Table 3). In general among diabetic rats, the

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activities of these antioxidant enzymes were decreased in brain regions. However with EU
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treatment, activity levels of these enzymes were enhanced differentially (Table 3). The SOD
activity in Ct and St were diminished and EU treatment normalized the same. Further, the
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activity levels of GR and TRR were reduced in all the brain regions of diabetic rats. While the
activity of phase II enzyme, GST was significantly diminished in Ct of diabetic rats, it was
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elevated with EU treatment. However, the enhanced activity of GST in St/ Hc regions of
diabetic rats remained unaffected with EU treatment. Although, activity levels of GPx were
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increased with EU treatment in Ct and Hc among diabetic rats, the activity of CAT was
differentially affected in all the brains regions among different groups (Table 3).
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3.2.6. Oxidative stress markers, MTT reduction and activities of mitochondrial enzymes
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Among untreated diabetic rats, the mitochondrial fractions of all the brain regions showed
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an increase in oxidative markers with a concomitant decrease in MTT reduction (Fig. 6 and
Fig. 7). EU treatment among diabetic rats caused a marked reduction in the levels of ROS and
PC (Fig. 6A and Fig. 7B). Further a similar effect was also observed in the levels of HP, NO
(Fig. 6B and C) and MDA (Fig. 7A). Among diabetic rats MTT reduction was enhanced
significantly with EU treatment (Fig. 7C).
In general the activities of complex I – III and SDH (Fig. 8A and B) were reduced among
the diabetic rats. EU supplementation significantly enhanced the activities of these enzymes in
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all brain regions examined. While the activity of CS was reduced significantly only in Ct and
St, its activity was enhanced in all the regions with EU supplementation (Fig. 8C).

3.2.7 Modulatory effect of EU on AChE activity

EU treatment significantly diminished the endogenous activity level of AChE in Ct, Cb

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and Hc (Fig. 9). Although an increase in AChE activity was significant in Ct and Cb, it was
marked (40%) in St among diabetic rats. Interestingly, EU treatment caused varying degree of

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reduction in the levels AChE in brain regions. However, no significant change was evident in
Hc among treated and untreated diabetic rats.

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4. Discussion

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The present study aimed to examine if EU, a well-known spice bioactive can be
efficacious as a therapeutic intervention agent under diabetic condition. To achieve this,
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initially we chose to assess the protective effect of EU in in vitro model under hyperglycemic
condition (100 mM glucose). The characteristics, culture conditions and usefulness of the
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human neuroblastoma cell line SH-SY5Y as a model for axonopathy has been well reviewed
(Forsby, 2011). Hyperglycemia is a risk factor for neuronal dysfunction especially impairment
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in signalling mechanisms (Guleria et al., 2006). Hyperglycemic conditions are known to

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generate ROS in both in vitro and in vivo situations in part through the formation and
downstream signalling effects of AGEs, polyol pathway and inflammatory responses leading
to apoptosis (Obrosova, 2009; Pazdro and Burgess, 2010; Negi et al., 2011). In the present
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study, glucose exposure to SHSY5Y cells resulted in enhanced levels of oxidative markers
with a concomitant reduction in the GSH levels. EU exposure rescued cells from glucose
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induced death as evidenced by an increase in cell survivability. Our salient findings such as
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enhancement in the oxidative markers viz., hydrogen peroxide, ROS as well as 3-NT levels
corroborate the previous findings in cell models (Hsu et al., 2013). Interestingly, EU treated
cells exhibited diminished levels of oxidative markers with concomitant increase in GSH
levels clearly suggesting its ability to up-regulate antioxidant defences in the cell model. This
observation is consistent with the previous reports on EU (Hidalgo and Rosa, 2009). Further,
cells exposed to either glucose or EU exhibited enhanced levels of heat shock proteins in
accordance with previous reports of activation of HSPs under stress conditions (Hooper and
Hooper, 2005). However, cells co-exposed to glucose and EU exhibited significantly
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diminished levels of HSP. There exists conflicting reports which describe both increase and
decrease in the levels of HSP in various diabetic models (Hooper and Hooper, 2005; Kamiya
et al., 2006). The vital role of HSP in tissue protection and repair mechanisms against
pathological conditions and pathology-dependent effects linked to small HSP expression has
been recently reviewed (Li and Dobrowsky, 2012; Al-Khatib, 2013).
EU, a well known antioxidant and anti-inflammatory molecule, has been extensively

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investigated for multiple functions in various models of diabetes. It has been attributed with
diverse roles with respect to its ability to affect multiple targets under diabetic condition in

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various vital organs (Mnafgui et al., 2013; Sanae et al., 2014; Srinivasan et al., 2014).
Recently, we have reported the neuroprotective effects EU in acrylamide induced neuropathy

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models. In the Drosophila model, EU rendered robust neuroprotection against acrylamide
induced locomotor deficits and biochemical alterations including oxidative stress markers
(Prasad and Muralidhara, 2012). Consistent with this, oral supplements of EU to acrylamide

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administered rats markedly improved the redox imbalance in sciatic nerve and brain regions
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with concomitant improvement in behavioural assessments for sensory and motor functions
(Prasad and Muralidhara, 2013). In a recent study, EU administration among diabetic rats was
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demonstrated to significantly reduce the levels of blood glucose and glycosylated hemoglobin
with concomitant increase in the plasma levels of insulin (Srinivasan et al., 2014). However,
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data on the propensity of EU to restore diabetes induced encephalopathy and neuropathy in

experimental models is rather limited. Hence, we aimed to examine the potential of EU to
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restore the pre-existing behavioural phenotype, oxidative stress in brain regions (and in sciatic
nerve, representative of the peripheral nervous system (PNS)) using an intervention approach.
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However, we have presented data only in the brain regions in this communication.
In the present model, diabetic rats exhibited characteristic neuropathic signs in terms of
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sensory and motor dysfunction (Data not shown). Consistent with previous reports, diabetic
rats displayed significant sensory deficits as early as 2 weeks and motor/ co-ordination
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deficits develop among significant number of rats only by 6 weeks (Zochodne et al., 2008;
Prasad and Muralidhara, 2013a; 2014). Hence, we chose to intervene the diabetes associated
neuropathology with EU administration from week 7 onwards. We continued the EU
treatment until significant improvement in behavioural assessments ensued which were
ascertained by increase in the latency period for sensory function and normalization of motor
function (Data not shown).
Earlier, we have reported the spectrum of diabetes associated behavioural impairments as
well as biochemical alterations in both CNS and PNS (Prasad and Muralidhara, 2014). In the
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present model, although EU treatment did not affect body weight gain and blood glucose
levels among non-diabetic rats, a significant improvement was evident among diabetic rats.
Consistent with previous reports we observed marked elevation in the oxidative markers in
the brain tissues of the diabetic rats (Negi et al., 2011; Prasad and Muralidhara, 2014).
Interestingly, EU treatment caused significant diminution in the levels of oxidative markers
within all brain the regions studied. This would have resulted probably from the up-regulation

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of GSH and TSH levels as evidenced by their enhanced levels among EU treated rats. This is
in line with earlier findings reported in different models (Baskaran et al., 2010; Prasad and

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Muralidhara, 2013). The blood glucose levels among EU treated diabetic rats were lowered
significantly (although not normalized) and is quite likely that the glucose flux to various

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pathways leading to the production of ROS/ RNS is lowered. While the activity of enzymes
such as GR and TRR were diminished among diabetic rats, EU treatment markedly enhanced
the activity levels suggesting its possible role in the maintenance of the antioxidant pool. We

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observed a differential response with respect to other enzymatic activities and the reason/s for
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such a response may be simply due to inherent differences in the detoxification processes in
different regions of the brain
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Mitochondrial dysfunction in neurons and Schwann cells among diabetic patients and
animal models of diabetes is well appreciated (Chowdhury et al., 2013). Although
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mitochondria are important for their role in generating the majority of cellular ATP via
electron transport chain, they are involved in other essential metabolic functions such as
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generation by the tricarboxylic acid (TCA) cycle metabolites, oxidative catabolism of amino
acids and urea cycle. Mitochondria not only generates ROS for important signalling functions,
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but also controls cytoplasmic calcium levels and synthesise important biomolecules such as
cellular Fe/S clusters and protein cofactors (reviewed by Patti and Corvera, 2010; Hernandez-
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Beltran et al., 2013). Hence, it is well accepted that, mitochondrial dysfunction (and
associated oxidative damage) is one of the primary factors in DN pathology (Fernyhough et
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al., 2010; Kamboj and Sandhir, 2011; Urban et al., 2011; Chowdhury et al., 2013). Consistent
with previous reports, we observed enhanced oxidative stress in the mitochondria among
different regions of the brain (Kamboj and Sandhir, 2011). Manifestation of oxidative stress
was revealed by the elevated levels of ROS, HP and NO. Further the important oxidized end
products of lipids (MDA) and proteins (PC) were also enhanced. Although, mitochondria of
EU treated diabetic rats exhibited marked diminution in the levels of oxidative stress markers,
it is not clear whether this is a direct implication of EU or it is due to its effects on
antioxidants present in the cytoplasm. Further some of the important enzymes of mitochondria
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exhibited enhanced activities among the EU treated diabetic rats clearly suggesting its ability
to attenuate mitochondrial dysfunction.
Mitochondria exhibit impaired calcium handling and elevated levels of ROS under
metabolic stress. Further, there is a close link between mitochondrial stress and associated
disruption in the cellular calcium homeostasis. This in turn is responsible for other
biochemical events such as activation of NOS which adds to the oxidative burden of the cell

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further hampering its normal function. Previous reports implicate calcium ion dys-regulation
and oxidative damage/ inflammatory response in degenerative pathology of DN (Latham et

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al., 2009; Naziroglu, 2011; Naziroglu et al., 2012). In the present model, the levels of
cytosolic calcium were also elevated in parallel to that of NO among the different

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experimental groups. Consistent with previous reports, brain regions of diabetic rats showed
decreased activities of mitochondrial enzymes (as well as MTT reduction) (Hu et al., 2009;
Kamboj and Sandhir, 2011). Interestingly the activities were restored with EU treatment

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clearly suggesting its potential to attenuate the mitochondrial dysfunctions. Further studies
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are required to decipher the underlying mechanisms for the modulatory effects exhibited by
EU.
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Previously, researchers have assessed cholinergic function with a view to understand the
learning and memory deficits under conditions of diabetic encephalopathy. AChE, is an
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important regulatory enzyme that degrades acetylcholine and inhibits its physiological role,
importantly synaptic transmission and conduction across cholinergic synapses (Schmatz et al.,
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2009). The involvement of significant alterations in neurotransmission as part of DN

pathology has been reported previously (Peeyush et al., 2010; Patil et al., 2014; Prasad and
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Muralidhara, 2014). In the present study, a marked increase in the AChE activity was evident
in Ct, Cb and St of diabetic rats. Increase in AChE activity leads to break-down of
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acetylcholine, which is needed for cognitive functions. Hence by lowering the activity of
AChE, the acetylcholine levels can be improved resulting in alleviation of cognitive
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dysfunction. Recently pioglitazone was found to improve cognitive deficits in part by

reducing the AChE activity in brain regions (Yin et al., 2013). Interestingly, the hippocampus,
located in the medial temporal lobe under the cortical region, responsible for consolidation of
short-term and long-term memory did not exhibit any change in the activity of AChE in the
present study. A plausible reason for this may be that the memory deficits develop only in
longer durations of experimental diabetes. However, EU treatment (control/ diabetic) resulted
in significant (yet differential) reduction in the AChE activity in all other brain regions tested.
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These data are in agreement with our previous reports on diabetes and effect of EU in
acrylamide model of neuropathy in rats (Prasad and Muralidhara, 2013, 2014)

5. Conclusions
In conclusion, we have obtained significant evidence which demonstrate the potential
neuromodulatory effect of EU in SHSY5Y cells and rat model of diabetes. EU treatment in

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cell model not only offered significant protection against hyperglycemia induced cell death,
enhanced GSH levels, but also attenuated oxidative stress. Further, its neurorestorative

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potential was exemplified in terms of its ability to abrogate pre-existing oxidative
impairments, mitochondrial dysfunctions and cholinergic deficit in different brain regions of

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STZ induced diabetic rats. Hence we propose that EU may be employed as a complementary
therapeutic molecule owing to its several positive attributes such as its ability to attenuate
oxidative/ inflammatory markers, modulate metabolic and neurotransmission activities as well

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as abrogate behavioural impairments associated with diabetes (Nangle et al., 2006; Mnafgui et
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al., 2013; Srinivasan et al., 2014). Based on our previous findings, we also propose its use
along with other molecules such as geraniol, a well-known monoterpene, in the management
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of diabetes associated complications in CNS and PNS.

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CONFLICT OF INTEREST
The authors declare no conflict of interest
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ACKNOWLEDGEMENTS
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We wish to thank the Director, CSIR-CFTRI, for his keen interest in this area of research.
Also, the grant from the Department of Science and Technology (Women Scientist Scheme
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WOS - A) New Delhi, Government of India (Ref: SR/ WOS-A/ LS-201/ 2008) to the first
author is greatly acknowledged.
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Legends for the figures

Fig. 1:

Effect of different concentrations of glucose on cell survivability (A) and the modulatory
effect of eugenol (EU – 5, 10 and 20 µM) on co-exposure with glucose (Glc, 100 mM) in

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SHSY5Y cells (B) for 24 h;
Values are mean ± SE (3 sets of experiments). Data analysed by one way analysis of variance

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(ANOVA) followed by Tukey’s test for comparison of means. * significant p ≤ 0.05 against
control and *** significant at p ≤ 0.001 against glucose (Glc, 100 mM)

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Fig. 2;

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Modulatory effect of eugenol (EU, 5 and 10 µM) on reactive oxygen species (A),
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hydroperoxides (B) and reduced glutathione (C) when co-exposed with glucose (Glc, 100
mM) in SHSY5Y cells for 24 h;
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Values are mean ± SE (3 sets of experiments). Data analysed by one way analysis of variance
(ANOVA) followed by Tukey’s test for comparison of means. * significant (p ≤ 0.05) against
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Control and # significant (p ≤ 0.05) against glucose (Glc, 100 mM)

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Fig. 3
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Modulatory effect of eugenol (EU, 5 and 10 µM) on the levels of HSP70 (A) and 3 -
nitrotyrosine (B) in SHSY5Y cells co-exposed with glucose (Glc, 100 mM) for 24 h as
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evidenced by slot blot analysis

Values are mean ± SE (3 sets of experiments). Data analysed by one way analysis of variance
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(ANOVA) followed by Tukey’s test for comparison of means. * significant p ≤ 0.05.

Fig. 4

Amelioration of elevated levels of oxidative markers- reactive oxygen species (A),

hydroperoxides (B) and nitric oxide (C) in cytosolic fractions of different brain regions of
diabetic rats by eugenol (EU) treatment in an intervention paradigm
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Values are mean ± SE (n=6). Data analysed by one way analysis of variance (ANOVA)
followed by Tukey’s test for comparison of means. *significant against control and # against
diabetic at p ≤ 0.05

Fig. 5

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Amelioration of elevated levels of malondialdehyde (A), protein carbonyls (B) and calcium
(C) in cytosolic fractions of different brain regions of diabetic rats by eugenol (EU) treatment

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in an intervention paradigm
Values are mean ± SE (n=6). Data analysed by one way analysis of variance (ANOVA)

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followed by Tukey’s test for comparison of means. *significant against control and # against
diabetic at p ≤ 0.05

Fig. 6
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Significant reduction in the levels of reactive oxygen species (A), hydroperoxides (B) and
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nitric oxide (C) in mitochondrial fractions of different brain regions of diabetic rats by
eugenol (EU) treatment in an intervention paradigm
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Values are mean ± SE (n=6). Data analysed by one way analysis of variance (ANOVA)
followed by Tukey’s test for comparison of means. *significant against control and # against
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diabetic at p ≤ 0.05
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Fig. 7
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Modulatory effect of eugenol (EU) treatment on the levels of malondialdehyde (A), protein
carbonyls (B) and extent of MTT reduction (C) in mitochondrial fractions of different brain
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regions of diabetic rats in an intervention paradigm

Values are mean ± SE (n=6). Data analysed by one way analysis of variance (ANOVA)
followed by Tukey’s test for comparison of means. *significant against control and # against
diabetic at p ≤ 0.05
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Fig. 8

Restorative effect of eugenol (EU) on the activities of mitochondrial marker enzymes-

Complex I – III (A) Succinate dehydrogenase (B) and Citrate synthase (C) in different brain
regions of the diabetic rats in an intervention paradigm
Values are mean ± SE (n=6). Data analysed by one way analysis of variance (ANOVA)

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followed by Tukey’s test for comparison of means. *significant against control and # against
diabetic at p ≤ 0.05

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Fig. 9

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Modulation in the activity of acetylcholinesterase from different brain regions of diabetic rats
employing an intervention treatment paradigm with eugenol (EU)

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diabetic at p ≤ 0.05
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Table 1:
Modulatory effect of eugenol (EU) treatment on blood glucose levels (mg/ dL) among
diabetic rats in an intervention model

Control EU Diabetic Diabetic + EU

6 weeks 94.0 ± 2 96.3 ± 3 465.8 ± 27* 465.8 ± 28*

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8 weeks 96.3 ± 2 95.2 ± 3 464.2 ± 20* 404.2 ± 30*

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10 weeks 96.0 ± 2 96.3 ± 2 475.8 ± 8* 324.2 ± 51*

12 weeks 96.0 ± 3 94.8 ± 3 462.5 ± 9* 235.8 ± 34*#

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Values are mean ± SE (n=6)

Data analyzed by one way analysis of variance (ANOVA) followed by Tukey’s test for

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comparison of means. * Significant against control; # against diabetic at p ≤ 0.05.
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Table 2

Modulatory effect of eugenol treatment on reduced glutathione (GSH) and total thiols (TSH)
in brain regions in diabetic rats in an intervention model

Enzymes/
Control EU Diabetic Diabetic + EU
Group

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Cortex
GSH1 5.80 ± 0.12*#

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5.05 ± 0.08 4.97 ± 0.16 3.66 ± 0.14*
TSH2 15.2 ± 0.2 15.1 ± 0.4 15.0 ± 0.4 21.0 ± 0.5*#

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Cerebellum
GSH1 4.98 ± 0.04 5.43 ± 0.08* 3.56 ± 0.15* 5.70 ± 0.11*#
TSH2 12.3 ± 0.5 19.9 ± 0.9* 14.6 ± 1.0 21.0 ± 0.5*#

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GSH1 4.98 ± 0.17 5.10 ± 0.05 3.59 ± 0.14* 5.37 ± 0.18#
TSH2 14.9 ± 0.2 13.9 ± 0.8 12.2 ± 0.3* 16.5 ± 0.6#

Hippocampus
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GSH1 4.91 ± 0.06 5.16 ± 0.24 3.34 ± 0.12* 5.89 ± 0.13*#

TSH2 16.2 ± 0.5 20.7 ± 2.0* 18.1 ± 0.6 25.2 ± 0.4*#
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Values are mean ± SE (n=10). Eugenol: EU, 10 mg/kg bw/ ip., alternate days 6 weeks, post 6
weeks of diabetes induction. Data analyzed by one way analysis of variance (ANOVA)
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followed by Tukey’s test for comparison of means. *significant against control and # against
diabetic at p ≤ 0.05)
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– Reduced Glutathione (ug/ mg protein)
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– Total thiols (nmol/ mg protein)
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Table 3

Effect of eugenol treatment on the activity levels of selected enzymes in cortex and
cerebellum of diabetic rats in an intervention model

Enzymes/
Control EU Diabetic Diabetic + EU
Group

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Cortex
GST1 43.2 ± 1.5 40.5 ± 0.5 29.1 ± 1.6* 47.7 ± 1.0 #
SOD2 63.1 ± 0.9 67.0 ± 1.2 55.2 ± 1.4* 66.4 ± 1.9 #

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CAT3 6.00 ± 0.08 7.65 ± 0.65* 7.54 ± 0.32* 5.77 ± 0.18 #
GPx4 29.7 ± 1.0 34.1 ± 2.7 21.5 ± 0.7* 37.1 ± 2.9 #
GR5 31.6 ± 1.0 47.3 ± 1.2 * 23.4 ± 1.2* 47.2 ± 2.8*#

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TRR6 6.01± 0.08 8.85 ± 0.28* 5.20 ± 0.11* 6.18 ±0.39

Cerebellum
GST1

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41.7 ± 0.7 42.8 ± 1.5 48.7 ± 2.9 49.1 ± 1.5
SOD2 67.5 ± 1.5 62.7 ± 1.3 72.9 ± 3.8 62.2 ± 7.0
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CAT3 6.05 ± 0.06 7.19 ± 0.35 7.30 ± 0.4 7.02 ± 0.28
GPx4 26.2 ± 1.1 27.6 ± 2.1 37.2 ± 1.3* 35.8 ± 1.4*
GR5 31.6 ± 0.5 29.5 ± 1.0 22.1 ± 1.5* 27.4 ± 0.6*#
TRR6 7.90 ± 0.08 10.8 ± 0.38 5.91 ± 0.40* 13.1 ± 0.17*#
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Values are mean ± SE (n=6);

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Eugenol: EU, 10 mg/kg bw/ ip., alternate days 6 wks, post 6 wks of diabetes induction.
Data analyzed by one way analysis of variance (ANOVA) followed by Tukey’s test for
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comparison of means. * significant against control; # against diabetic at p ≤ 0.05

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– Glutathione-S-transferase, nmol conjugate formed/ min/ mg protein
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– Superoxide dismutase, U/ mg protein
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– Catalase, nmol hydrogen peroxide decomposed/ min/ mg protein
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– Glutathione peroxidase, nmolNADPH oxidized/ min/ mg protein
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– Glutathione Reductase, nmolNADPH oxidized/ min/ mg protein
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– Thioredoxin Reductase, nmol substrate/ min/ mg protein
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Table 4

Effect of eugenol treatment on the activity levels of selected enzymes in striatum and
hippocampus of diabetic rats in an intervention model

Enzymes/
Control EU Diabetic Diabetic + EU
Group

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Striatum
GST1 44.9 ± 1.3 48 ± 1.0 57.1 ± 0.7* 56.9 ± 3.2*
SOD2 28.8 ± 1.1 26.3 ± 1.0 9.17 ± 0.8* 26.9 ± 0.6 #
CAT3 2.61 ± 0.04*#

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3.86 ± 0.04 3.18 ± 0.21* 1.96 ± 0.12*
GPx4 17.9 ± 0.3 25.2 ± 0.9* 31.1 ± 1.2* 29.9 ± 2.0*
GR5 11.5 ± 0.99 11.4 ± 0.34 4.80 ± 0.31* 8.82 ± 1.01 #

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TRR6 5.35 ± 0.10 5.52 ± 0.06 1.43 ± 0.17* 4.81 ± 0.59 #

Hippocampus
GST1 40.0 ± 0.7 46.1 ± 2.3 58.9 ± 3.5* 58.4 ± 2.5*

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SOD2 30.8 ± 0.7 30.1 ± 0.8 43.8 ± 6.6* 29.3 ± 1.8 #
CAT3 ND ND ND ND
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GPx4 31.7 ± 1.9 34.3 ± 1.1 26.1 ± 0.8* 34.7 ± 1.3 #
GR5 11.0 ± 0.4 10.7 ± 1.26 4.91 ± 0.70* 13.5 ± 0.91 #
TRR6 4.95 ± 0.11 5.34 ± 0.28 3.21 ± 0.33* 4.10 ± 0.28
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Values are mean ± SE (n=6);

Eugenol: EU, 10 mg/kg bw/ ip., alternate days 6 wks, post 6 wks of diabetes induction.
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ND: Not Determined.

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Data analyzed by one way analysis of variance (ANOVA) followed by Tukey’s test for
comparison of means. * significant against control; # against diabetic at p ≤ 0.05

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– Glutathione-S-transferase, nmol conjugate formed/ min/ mg protein

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– Superoxide dismutase, U/ mg protein
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– Catalase, nmol hydrogen peroxide decomposed/ min/ mg protein
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– Glutathione peroxidase, nmolNADPH oxidized/ min/ mg protein

5
– Glutathione Reductase, nmolNADPH oxidized/ min/ mg protein
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6
– Thioredoxin Reductase, nmol substrate/ min/ mg protein
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Highlights

Eugenol (EU) coexposure t with glucose (100 mM) enhanced survivability of SHSY5Y cells

EU attenuated glucose induced oxidative stress(OS) markers

In an intervention paradigm, EU treament diabetic rats, significantly diminished OS markers

in cytosolic and mitochondria of different brain regions

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EU enhanced the activities of various redox enzymes and mitochondrial enzymes

EU differentially restored the elevated activity of acetylcholinesterase in brain regions

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