Scavenging and Antioxidant Activity of Aqueous Plant Extracts towards Superoxide Radical Anion

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


2/17

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]


[Rhoeo spathacea Extract]

y=0.0153x+10.141

50=0.0153x+10.141

IC50=(50-10.141)/0.0153

*IC50= 2,605ug/mL



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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]


[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



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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.

Documents

Scavenging and Antioxidant Activity of Aqueous Plant Extracts towards Superoxide Radical Anion