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By:Nasreen Ahmad
12PGD538Adv. PG Diploma in Nanotechnology
Centre of Excellence in Materials Science (Nanomaterials)
Dept. of Applied Physics, AMU, Aligarh.
PtNH3
ClNH3
Cl
H
HHH H
H
Mitigation of Cisplatin induced toxicities by Nanocurcumin in
male wistar rats
Under Supervision of:Prof. Alim Hussain NaqviCo-ordinator,Centre of Excellence in Materials Science (Nanomaterials)Dept. of Applied Physics, AMU, Aligarh.
Under co-supervision of: Prof. M. Mobarak Hossain
Director, Interdisciplinary Brain Research Centre,
Faculty of Medicine,AMU, Aligarh.
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Plan of Talk
What is Cisplatin?
What is Nanocurcumin?
How to synthesize nanocurcumin?
Is it really Nanocurcumin?
How did we do it?- Experimental design.
Is it really effective?-our findings.
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Pt
NH3
ClNH3
Cl
H
HHH H
HCisplatin
It is a chemotherapeutic drug.
[molecular formula- H6Cl2N2Pt, molar mass-301.1g/mol]
It is used to treat various types of cancers.
It is accompanied by various toxicity including, Neurotoxicity, Genotoxicity, Ototoxicity, Nephrotoxicity and Hepatotoxicity
Image showing enlarged rat stomach on cisplatin exposure
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Pt
NH3
ClNH3
Cl
H
HHH H
H Hepatocellular carcinoma in rats
H
HHH H
H
ATPADP
Pt
NH 3
ClNH3
Cl
H
HHH H
H
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Adenine Guanine Cytosine Thymine
H
HH HHH
Pt
N N
H
H
H H
H
H
NN
H
H
HH H
H
2Cl
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Adenine
Guanine
Cytosine Thymine
Pt
N NH
H
H H
H
H
NN
APOPTOSIS
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H3CO
H3C
OH
OH
OH
O
Nanocurcumin (diferuloylmethane)
antioxidant, anti-inflammatory, antimicrobial
hepato and nephro-protective , myocardial infarction protection
A natural product extracted from curcuma longa
Soluble in water while curcumin was not soluble.
More bioavailability due to small sizes.
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Synthesis of NanocurcuminDried Turmeric Ethanol:water::4:1
Shake vigorously for 10-15 min to obtain the shown image
Left on standing overnight.
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{The upper clear liquid was collected and this process was repeated until a clear solution was obtained}
(dropwise in to water at 95 ºC)
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(Nanocurcumin)
Dried &stored for further characterisation
Kept in liquid form for oral dosing to subject rats.
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Experimental Design
Brain taken for Biochemical & Protein Estimations Blood Sample
collected for Hematological Estimations
Femur (Bone marrow) collected for Genotoxicity Estimations
Epididymis collected for the assessment of Sperm Abnormalities
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Experimental Procedures
TISSUE IS HOMOGENIZED TO MAKE 10 % HOMOGENATE
(W/V)
POST MITOCHONDRIAL SUPERNATANT (PMS) IS PREPARED BY CENTRIFUGATION AT 10000rpm
FOR 20 MINUTES
CONTROL
Cisplatin GRP
Nanocurcumin GRP 1 (0.5mg/kg)
Treatment GRP 2(NC-1+cis)
Rat Brain
Nanocurcumin GRP 2 (1mg/kg)
Treatment GRP 1(NC-0.5+cis)
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Assessment Parameters
Non-Enzymatic assays
• Non-Protein thiol• GSH Assay• TSH Assay
Biochemical Estimation
Lipid Peroxidation
Antioxidant Enzyme Assay Glytathion-S-
Transferase estimation
Reproductive toxcity
Hematological Estimations
Genotoxicity Estimations
Micronucleus Test
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UV-Vis Spectroscopy
Absorbance maxima at 420nm
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Fourier Transmission Infrared Microscopy
peaks at 3415 cm−1
corresponds to OH stretching vibrations
at 1,608 cm−1 were attributed to carboxylate OCO asymmetric stretching
at 1,588 cm−1 was assigned to C–OH deformation vibration with contribution of O–C–O symmetric stretching vibration of carboxylate group
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(A)Figure shows the TGA of nanocurcumin and fig (B)shows the DSC of nanocurcumin
Differential Scanning Calorimetry & Thermogravimetric Analysis
first phase transition at 100.934ºC and the second phase transition at 440.699ºC
air dehydrated from 49 to 140 °C and the free curcumin in the product melted at ~ 250 °C
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TEM images show sizes in the range of 10-20nm.
Transmission Electron Microscopy
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GSH
Effect of different concentration of Nanocurcumin on cisplatin altered GSH level in brain of Male Wistarrats.Values are expressed as mean ± standard error (SE, n=6). It is measured as µmoles GSH/gm tissue. Significant difference are indicated by (*)when compared with control and (#) when compared with cisplatin group.
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Effect of different concentration of Nanocurcumin on cisplatin induced depletion of non-protein thiols in brain of Male Wistarrats.Values are expressed as mean ± standard error (SE, n=6). It is measured as µmoles NP-SH/gmtissue.Significant difference are indicated by (*)when compared with control and (#) when compared with cisplatin group.
Non-Protein Thiol
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Total ThiolEffect of different concentration of Nanocurcumin on cisplatin altered T-SH level in brain of Male Wistarrats.Values are expressed as mean ± standard error (SE, n=6). It is measured as mmoles TSH/gm tissue. Significant difference are indicated by (*)when compared with control and (#) when compared with cisplatin group
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Lipid Peroxidation
Effect of different concentration of Nanocurcumin on cisplatin induced lipid peroxidation (LPO) in brain of Male Wistarrats.Values are expressed as mean ± standard error (SE, n=6). LPO is measured as µmoles TBARS formed/hr/gm tissue.
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Glutathion-S-Transferase
Effect of different concentration of Nanocurcumin on cisplatin altered GST activity in brain of Male Wistarrats.Values are expressed as mean ± standard error (SE, n=6). It is measured as nmoles of CDNB conjugated/min/mg protein.
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(E)
(D)(C)
(F)
(A) (B)
Micronucleus Test
(A) Represent normal bone marrow cells while (B) shows the micronucleated cells, (C&D) shows the normal cells,and (E&F)shows decline in MNs count with protection of nanocurcumin.
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Dose Dependent Comparative representation of Micronucleus
Dose-dependent profile of Micronucleated Polychromatic Erythrocytes by Cisplatin and Nanocurcumin in Rattus norvegicus.
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Total Sperm Abnormality
Fig (A) showing the normal sperm, (B,C,D) coiled tail, amorphous head, broken head, reversed head position, and coiled sperms showing the abnormality caused by the cisplatin.
(A) (B) (C)
(D)
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Dose Dependent Comparative representation of Sperm Abnormalities
Frequency distribution of sperm abnormalities in cisplatin and Nanocurcumin treated R. norvegicus
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Hematological Assessments
(A, C& D) showing the normal blood cells and (B,E&F) shows various kind of anomalies in blood cells of rat by the treatment of cisplatin.
(A) (B)
(C) (D)
(F)(E)
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Inference From This Study?? Small sizes of Nanocurcumin enhanced its bioavailability.
Proved to be a potent candidate for the management of cisplatin
related side effects.
And Much More… Further we are also investigating liver, kidney and heart samples for
different analysis.
Also samples are being investigated for histopathological effects.
Comet assays are also being done.
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