Post on 14-Apr-2018
7/30/2019 Scavenging and Antioxidant Activity of Aqueous Plant Extracts towards Superoxide Radical Anion
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Scavenging and Antioxidant Activity of
Aqueous Plant Extracts towards Superoxide
Radical Anion
Wanda Figueroa Cuilan
Mentor:
Jannette Gavilln-Surez, Ph.D
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Reactive Oxygen Species (ROS)
Reactive Oxygen Species
How are the radicals are
formed?
Antioxidants
Oxidative Stress Figure 1. Antioxidant molecule donating an electronto the radical atom
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Diabetes
Diabetes mellitus is a disorder in which blood sugar (glucose)levels are abnormally high.
There are two types of Diabetes:
Type 1: results from the body's failure to produce
insulin.
Type 2: the body does not respond correctly to
insulin.
insulin resistance
means that adipose tissue, liver, and muscle
(fibers) cells do not respond normally to
insulin
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Relevance of this Research
Diabetes and obesity are also associated with theoverproduction of mitochondrial reactive oxygen species(Superoxide), leading to mitochondrial and cellularoxidative damage. This, in turn, contributes to the
development and progression of diabetic complications andto worsening of the diabetic state per se. William Sivitz,MD, Endocrinologist
Goal
Study superoxide radical scavenging and antioxidant activityof plants used as diabetes adjuvants in traditional medicine.
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Objetives
Tapeinochil
us
ananassaehttp://www.tropicalflowe
rfarm.com:80/imagepopu
p.html
Rhoeospathaceahorticulture.missouri.ed
u
Costus sp.
www.efloras.org
Syzygiumjambos
www.subtropical.co.nz
Determine the scavenging activity of plant
extracts as Costus sp., S.jambos, T.anassae, y
R.spathacea towards the Superoxide radical
anion.
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Research Design
Negative Control,Positive Control or
Extracts
10uL DMSO, 10uLQuercetine or 10uL Extracts
85uL Phosphate Buffer
25uL NBT
5uL PMS
25uL NADH
Blanc of theControl
10uL DMSO, 10uLQuercetine or 10uL Extracts
110uL Phosphate Buffer
25uL NBT
5uL PMS
Blanc NADH
125uL Phosphate
Buffer
25 NADH
Figure 1. Elisa Plates (96 cells, 200uL volume)
Blanc of NADH
Negative Control
Blanc of Negative Control
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
H
I
Methods: Testing the scavenging activity of Aqueous Extracts
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Abs Control(590nm) Abs Extract(590nm)
Abs Control(590nm)% I =
Research Design
1. Inhibitory Activity
2. Plot linear Regression
%I=m[S] + b
3. Calculate IC50
50=mx + b
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Results: Quercetine assay
Positive Control
y = 0.9811x + 3.8801
R = 0.8737
0
20
40
60
80
100
120
0 20 40 60 80 100 120
InhibitionP
ercentage(%)
[Quercetine ug/mL]
Inhibition Percentage (%) Vs. Quercetine
[Quercetine] Inhibition (%) Percentage
100ug/mL 92%
50ug/mL 78%
25ug/mL 21%
6.25ug/mL 6%
y=0.9811x+3.8801
50=0.9811x+3.8801
IC50= (50-3.8801)/0.9811
IC50= 47.088ug/mL
IC50 Calculation
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Results Costus sp.assay
Extract Analysis
1,072ug/mL Costus Sp.
Cualitative Results
y=0.035x-2.2969
50=0.035x-2.2969
IC50=(50+2.2969)/0.035
*IC50= 1,494.2ug/mL
*Preliminary Results
[Costus sp.] Inhibition (%) Percentage
1,072ug/mL 33%
667ug/mL 25%
533ug/mL 18%
333ug/mL 6.3%
IC50 Calculation
y = 0.035x - 2.2969
R = 0.9121
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
0 200 400 600 800 1000 1200
InhbitionPercent
age(%)
[Costus sp. Extract]
Inhibition Percentage (%) Vs.
[Costus sp. Extract]
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Results: Rhoeo spathacea
Extract Analysis
[Rhoeo Spathacea] Inhibition (%) Percentage
1,340ug/mL 31%
1,072ug/mL 26%
667ug/mL 21%
553ug/mL 18%
IC50 Calculation
[1,340ug/mL]
Cualitative Results
y = 0.0153x + 10.141
R = 0.9905
0
5
10
15
20
25
30
35
0 200 400 600 800 1000 1200 1400 1600InhibitionPercentage(%)
[Rhoeo Spathacea Extract ug/mL]
Inhibition Percentage (%) Vs.
[Rhoeo spathacea Extract]
y=0.0153x+10.141
50=0.0153x+10.141
IC50=(50-10.141)/0.0153
*IC50= 2,605ug/mL
*Preliminary Results
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Results:Tappeinochilus anassae
Extract Analysis
y = 0.0548x + 68.467
R = 0.9992
74
76
78
80
82
84
86
88
90
0 50 100 150 200 250 300 350 400
InhibitionPerce
ntage(%)
[Tappeinochilus Anassae Extract ug/mL]
Inhibition Percentage (%) Vs.
[Tappeinochilus anassae Extract]
[Rhoeo Spathacea] Inhibition (%) Percentage
373/mL 89%
233ug/mL 81%
117ug/mL 75%
IC50 Calculation
y=0.0548x+68.467
50=0.0548x+68.467
IC50=(50-68.487)/0.0548
[373 ug/mL]
Cualitative Results
*Preliminary Results
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Experimental Relevance of this research
We found a reproducible method that recreates the
superoxide radical environment.
We develop this method in Elisa 96 plates, reading
the absorbance in a microplate reader, according tothe standard absorbance of the apparatus.
Tendencies in inhibition of plant extracts are:
T. anassae > Cotus sp.> R.sphathacea
Superoxide Radical
Envinronment
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Future Work
Determine the scavenging activity aqueous
extracts towards Superoxide Radical Anion,
increasing and decreasing concentrations. Analyze methanolic plant extracts and their
respective IC50.
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References
Tomasz J. Guzik, MD, PhD. Mechanisms of Increased Vascular
Superoxide Production in Human Diabetes Mellitus Role of
NAD(P)H Oxidase and Endothelial Nitric Oxide Synthase, 2002
Francesco Cosentino, MD PhD, High Glucose Increases Nitric
Oxide Synthase Expression and Superoxide Anion Generation in
Human Aortic Endothelial Cells, 2002
William Sivitz, MD, Mitochondrial Dysfunction in Obesity and
Diabetes, US Endocrinology, 2010;6:20-7
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Acknowledgements
I would like to thanks Dr. Jannette Gavilln-
Surez as my mentor and lab Principal
Investigator (P.I.)
Collaborators as Biology Department and lab
technicians Nivea Franco and Angie Castell.
Special thanks to Instituto de Investigaciones
Interdiciplinarias of the University of Puerto
Rico at Cayey.