1. Effect of Zinc Oxide Nanoparticles Synthesized Using
Onion Bulb (Allium cepa) on Triton WR-1339 Induced
Hyperlipidemia in Rats
By
ADAMU, Saminu Shehu
(M.Tech./BCH/18/0170)
Supervisor: Dr H. A. Umaru
2. Introduction
• Hyperlipidemia is an umbrella term that refers to any of several acquired or genetic
disorder that result in a high level of lipid (fats, cholesterol and triglyceride).
• These lipids can enter the walls of arteries and increase the risk of hardening of the
arteries (artherosclerosis) which can lead to stroke and heart attack (Kang et al., 2009).
• This medical condition or problem is divided into two subtypes: primary hyperlipidemia
and secondary hyperlipidemia (Onwe et al., 2015).
3. Introduction cont’d
Primary Hyperlipidemia
• This usually take place as a result of genetic problems i.e., mutation within receptor protein.
• Thus, may be due to single (monogonic) gene defect or multiple (polygenic) gene defect (Joseph,
2005).
Secondary hyperlipidemia
• This arises as a result of other underlining diseases like diabetes, nephritic syndrome, chronic
alcoholism and use of drugs like corticosteroids and oral contraceptives (Joseph, 2005).
4. Introduction cont’d
Risk Factors
Non Modifiable Risk Factors
• Age: Because body’s chemistry changes with age. Thus, the risk of high cholesterol climbs.
Example, liver becomes less able to remove LDL cholesterol with age.
• Gender: Overproduction and defective clearance of the cholesterol, TG and LDL is the result of
the mutations of single or multiple genes (Weinreich and Frishman, 2014).
• Chronic Disease: High blood sugar contributes to higher levels of a dangerous cholesterol called
LDL and lower HDL which damage the lining of arteries (Saadi et al., 2007).
5. Introduction cont’d
Modifiable Risk Factors
• Medications: Drugs like thiazides, retinoids, estrogens and glucocorticoids, among others also
increase the risk of dyslipidemia (Jackson et al., 2002).
• Nutrition: Saturated fat found in animal products and trans fat found in some commercially baked
cookies can raise cholesterol level (Weinreich and Frishman, 2014).
• Lack of Exercise: Exercise helps boost HDL “good” while increasing the size of the particles that
make up LDL “bad” which makes it less harmful (Bener et al., 2009).
• Smoking: cigarette smoking damages the walls of blood vessels, making them more prone to
accumulate fatty deposits and thus lower HDL (Shattat, 2014).
6. Prevalence
• According to American Heart Association, the Centers for Disease Control and Prevention and
other government sources, cardiovascular disease is the leading global cause of death, accounting
for more than 17.3 million deaths per year, a number that is expected to grow to more than 23.6
million by 2030 (Niharika, 2017).
• Reports on the pattern of lipid profile abnormalities in blacks are scanty and inconsistent.
• This has greatly affected lipid lowering intervention using drugs and even life style changes, as an
attempt to provide a general picture of lipid pattern in sub-Sahara, especially Nigerians (Akuyam
et al., 2010).
7. Nanotechnology
• Recently, nanotechnology is one of the most active subjects of research and hence metal
nanoparticles have a great scientific interest because of their unique properties with applications
in divers areas.
• Nanotechnology refers to an emerging field with its applications in science and technology
research for the purpose of manufacturing new materials at nano scales in 1 to 100 nanometers
range (Albrecht et al., 2006).
• Nanomedicine has emerged as a rapidly growing field and proves to be a potent and effective
therapy against various diseases.
8. Zinc oxide Nanoparticles
• Zinc oxide nanoparticles (ZnO NPs), as one of the most important metal oxide nanoparticles
which are popularly employed in various industrial and biomedical fields (Smijs and Pavel, 2011).
• In this study, Zinc oxide nanoparticles will be used to investigate it is potential anti-hyperlipidemic
activity against Triton WR-1339 induced hyperlipidemia in rats.
• Therefore, the plant that will be carried out to mediate the synthesis of zinc oxide nanoparticles
(ZnO NPs) is using onion bulb extract of Allium cepa as a reducing agent.
• The optimized ZnO NPs thus obtained will be quantified and characterized using X-Ray diffraction
(XRD), Fourier Transform Infrared Spectroscopy (FT-IR) and Scanning Electron Microscopy.
9. Statement of the Problem
• Hyperlipidemia is a factor for atherosclerosis and related cardiovascular diseases, including
coronary heart disease, myocardial infarction and renal failure which are becoming a major health
problem in the world today (Xu et al., 2014).
• There are many drugs which are in use as hypolipidemic agent but the therapy is not cost-
effective.
• Currently available hyperlipidemic drugs have been associated with a number of side effects and
are unable to increase HDL levels (Karam et al., 2017).
• It is therefore a need for alternative treatment options that are less toxic and less expensive,
which can provide better safety and efficacy on a long term usage.
10. Aim and Objectives of the Study
Aim
• The aim of this study is to determine the effect of Zinc oxide nanoparticles from Onion bulb
extract on Triton WR-1339 induced hyperlipidemia in rats.
Objectives of the Study
• To determine the levels of Zinc oxide nanoparticles using Onion bulb.
• To determine the in vitro scavenging activity of Zinc oxide nanoparticles from Onion bulb using
DPPH and TBARS.
11. Objectives of the Study cont’d
• To determine the effect of Zinc oxide nanoparticles from Onion bulb on lipid profile parameters in
triton WR-1339 induced hyperlipidemic rats (TC, TG, LDL, HDL and AI).
• To determine the effect of Zinc oxide nanoparticles from Onion bulb on liver enzymes in triton
WR-1339 induced hyperlipidemic rats (ALT, AST and ALP).
• To determine the effect of Zinc oxide nanoparticles from Onion bulb on antioxidant enzyme
markers (Superoxide dismutase, Catalase and Glutathione peroxidase).
• To determine the effect of Zinc oxide nanoparticles from Onion bulb on cardiovascular
parameters (Troponin-I, Creatine kinase and Myoglobin).
• To determine the effect of Zinc oxide nanoparticles from Onion bulb on structural alterations
through histopathology of the heart.
12. Materials and Method
Materials
Equipments
• Spectrophotometer, Centrifuge, Homogenizer, Weighing balance, Thermometer.
Chemicals
• Triton WR-1339, Zinc nitrate, Normal Saline, DPPH, TBARS.
Reagents
• Lipid profile (), liver function (), Antioxidants enzymes kits (SOD, Catalase and GPx) and
Cardiovascular markers (Troponin-I, CK-MB and Myoglobin) reagent kits.
13. Materials cont’n
Plant Materials
• Fresh onion bulb (Allium cepa) will be purchased from Jaga-jaga local market. Identification and
authentication will be done in the herbarium of the department of Botany, school of life science,
Modibbo Adama University of Technology, Yola.
Experimental Animals
• Forty two adult male Wister rats weighing 100±10g will be used for this study. The rats will be
purchased from Veterinary research institute, Vom-Jos, Plateau state.
• The animals will be fed with standard pellet diet and water ad libitum for a period of two weeks
before experiment.
14. Preparation and Synthesis
Preparation of Onion Bulbs Extract
• Onion bulb will be rinsed with sterile dH2O and the outer casing will be removed while the fleshy
part will be washed with sterile dH2O. About 10gm of bulb will be grounded with a small volume
of dH2O. The extract will serve as reducing agent and stabilizer (Wu et al., 2019).
Synthesis of Zinc Oxide Nanoparticles
• The reaction mixture will be prepared by mixing of 10 ml of onion extract and 90 ml of 1mM zinc
nitrate in a 250ml conical flask and the mixture will be incubated in a dark condition at 37oC
followed by centrifugation at 10000rpm for 20 mins. The pellet will be taken and air dried
(Tensingh and Lega, 2018).
15. Characterization of ZnO NPs
Characterization of Zinc Oxide Nanoparticles
X-ray Diffraction (XRD) Analysis
• X-ray diffraction (Model PW1050/37 Philips) analysis will be used to determine the shape and
crystal structure of the synthesized ZnO NPs (Wu et al., 2019).
Fourier Transform Infrared Spectroscopy (FTIR)
• FTIR (FTIR-8400S SHIMADZU) analysis will be used to determine the chemical composition of the
synthesized ZnO NPs (Prathra et al., 2011).
Scanning Electron Microscopy (SEM)
• SEM (JEOL JSM-6480 LV) analysis will be used to assess the particle surface size and size
distribution (Forough and Farhadi, 2010).
16. In vitro Antioxidant Activity of ZnO NPs
DPPH
• Method: The radical scavenging activity of ZnO-NPs will be determined by using DPPH assay
according to Chang et al. (2001). The decrease in the absorption of the DPPH solution after the
addition of ZnO-NPs will be measured at 517nm. Ascorbic acid (10mg/ml DMSO) will be used as
reference.
TBARS
• Method: The radical scavenging activity of ZnO-NPs will be determined by using TBARS assay as
described by Satoh, (1978) while the absorbance will be measured at 535nm.
Induction of Hyperlipidemia
• The rats will be induced by intraperitoneal injection of freshly prepared solution of Triton WR-
1339 (50mg/kg) in physiological solution in a single dose/day for a period of 14 days after fasting
for 5 hours (Levine and Saltzman, 2007).
17. Experimental Design
This experiment will be
conducted with 42 healthy
male Wistar rats at the
laboratory of the department
of Biochemistry, Modibbo
Adama University, Yola.
Experimental animals will be handle in accordance with the
international guiding principles of Biochemical research involving
animals (CIOMS, 1985). 42
Male Wistar
Rats
Group I
(6 Rats)
Normal control
(Standard diet)
for 28 days.
Group II
Hyperlipidemi
c control
[Triton WR-
1339 (50 mg /
kg BW/day)]
14 days +
standard diet
for 28 days.
Group III
Standard
[Triton WR-
1339
(50mg/kg
BW/day) for
14 days +
standard diet
for 28 days +
(Atorvastatin
10 mg / kg
BW/day) for
14 days
Group IV
Test group 1
[Triton WR-
1339 (50
mg/kg
BW/day)] for
14 days + ZnO
NPs (50mg/
kg BW/day)
for 14 days
Group V
Test group 2
[Triton WR-
1339 (50
mg/kg
BW/day)]
for 14 days +
ZnO NPs
(100 mg/ kg
BW/day) for
14 days.
Group VI
Test group 3
[Triton WR-
1339 (50
mg/kg
BW/day)] for
14 days +
ZnO NPs
(150mg/ kg
BW/day) for
14 days.
18. Biochemical and Histopathological
Parameters
Blood and Sample Collection
Within 28 days of treatment, 2 rats from each group will be sacrificed by cardiac puncture under
aneasthesia on the 1st, 3rd and 5th day, blood will be collected in to sample bottles. Serum will be
separated from the blood by centrifugation at 10,000rpm for 10 minutes and stored room
temperature until analysis. Heart will be collected for histopathological analysis.
Blood and
Organs of Rats
Histopathological
Analysis
Biochemical
Analysis
19. Determination of Biochemical Parameters
.
Antioxidant
Enzymes
Biochemical
Parameters
Lipid Profile Liver Enzymes
Cardiovascular
Parameters
TC, TG, LDL,
HDL and AI
ALT, AST and
ALP
SOD, CAT and
GPx
Troponin-I,
Creatine kinase
and Myoglobin
20. Methodology
Lipid Profile
Determination of Serum Total Cholesterol (TC)
• Method: Determination of total cholesterol will be based on CHOD-PAP enzyme colorimetric
method (Tietz, 1999) and (Young, 1999).
Determination of Serum Triglycerides
• Method: Determination of serum triglycerides will be based on the GPO/PAP enzyme colorimetric
method which involves lipase catalyzed hydrolysis (Trinder, 1969; Bucolo and David, 1973; Fossati
and Prencipe, 1982).
21. Methodology Cont’n
Determination of Serum High Density Lipoprotein (HDL) Cholesterol
• Method: Determination of serum HDL will be based on the PEG/CHOD-PAP colorimetric method
(Trider, 1969; Allain et al., 1974; Flegg, 1972; Grillo et al., 1981; Demacker et al., 1980).
Evaluation of Atherogenic Index
• Method: Atherogenic Index will be determine according to the method explained by Nwaghu,
(2010).
22. Methodology Cont’n
Liver Enzymes
Determination of Serum Aspartate Aminotransaminase (AST)
• Method: Serum AST concentration will be determined by the colorimetric method as
described by IFCC method (Bergmeyer et al., 1976).
Determination of Serum Alanine Aminotransaminase (ALT)
• Method: Serum ALT concentration will be determined by the method as described by
Raitman and Frankel's method (Zilva, 1979; Young, 1990).
Determination of Serum Alkaline Phosphatase (ALP)
• Method: Serum ALP concentration will be determined by the method as described by
pNPP Kinetic method (Bowers and McCommb, 1972; Recommendations for the German
Society for Clinical Chemistry, 1972).
23. Methodology Cont’n
Cardiovascular Parameters
Determination of Cardiac Troponin-i
• Method: Cardiac Troponin-i (cT-i) will also be quantitatively determine base on the fluorescence
Immunoassay (FIA) for the cT-i activity in serum using sandwich immunodetection method with
ichroma™ test instrument (Panteghini et al., 2004; Jillan et al., 2002).
Determination of Creatine Kinase (CK-MB)
• Method: CK-MB will be quantitatively determine base on the fluorescence immunoassay (FIA) for
the Creatine Kinase Isoenzyme-MB (CK-MB) activity in serum using sandwich immunodetection
method with ichroma™ test instrument (Bedside Multimarker Testing for Risk Stratification in
Chest Pain Units, 2001).
Determination of Myoglobin
• Method: Myoglobin will also be quantitatively determine base on the fluorescence Immunoassay
(FIA) for the myoglobin activity in serum using sandwich immunodetection method with
ichroma™ test instrument (Ordway and Garry, 2004; Lewandrowski, 2002)
24. Methodology Cont’n
Antioxidant Enzymes
Determination of Serum Superoxide Dismutase (SOD)
• Method: Serum SOD can be determine by colorimetric method as describe by
Nishikimi et al., (1972).
Determination of Serum Catalase Activity
• Method: Serum Catalase can be determine by colorimetric method as describe
by (Aebi, 1984).
Determination of Glutathione Peroxidase (GPx) Activity
• Method: Serum Glutathione Peroxidase can be determine using Ultra-Violet (UV)
method as describe by (Paglia and Valentine, 1967).
25. Methodology Cont’n
Determination of Histopathology of the Heart
Specimens of the heart tissues will be fixed immediately after removed in 10% formalin, and then treated with
conventional grade of alcohol and xylol embedded in paraffin, sectioned at about 5μ thickness. The sections will
be stained with hematoxylin and eosin (H&E) stain for studying the histopathological changes. Histological
slides would be examine under a light microscope at ×400 magnification. (Lillie, 1965).
Histopathological
Analysis
Heart of the
Rats
26. Statistical Analysis
• All numerical data that will be obtained from the study will be reported as Mean ± Standard error
of mean (S.E.M.). Statistical analysis will done using the Statistical Parkage for Social Science
(SPSS) version 24.0 (SPSS, Incorporation Chicago Illinois, USA). Differences between and within
group means will be analyzed using One-way analysis of variance (ANOVA) followed by T-test.
𝑝 < 0.05 will be considered as statistically significant.
