Research Experience:

“Scavenging and Antioxidant Activity Towards Superoxide Radical of Plant Extracts used as

Diabetes Adjuvant”

University of Puerto Rico at Cayey

Cayey Puerto Rico 00736

Wanda M. Figueroa-Cuilan

Special thanks to:

Aknowledgements

Aknowledgements

Dr. Jannette Gavillán-Suárez

“Lighthouses are our guides when

we are lost in the darkness of the

night”

Dr. Mariluz Franco

Dr. Jessica Gaspar

To investigate the in vitro biological activities

and in vivo efficacy of herbal formulations used as

diabetes adjuvants in Puerto Rico to develop new

therapeutic methods to treat diabetes.

Program Goal

Researches Around the world

Around the world several studies have been

conducted to discover good antioxidant and scavenging

plants extracts, in order to develop new therapeutics to

treat diabetes.

Example: Triphala (2009)

Plant Extract IC50 (ug/mL) Country Research Title

Spondias pinnata 13.46 India Antioxidant and free radical activity of Spondias pinatta

Ervatamia coronaria Stapf. 118.77 India Antioxidant and Free radical scavenging Activities of

Evartamia coronaria Stapf.

Panax ginseng 3.18 India In vitro evaluation of free radical scavenging activities of

Panax ginseng and lagertoemia speciosa

Lagerstroemia speciosa 6.15 India In vitro evaluation of free radical scavenging activities of

Panax ginseng and lagertoemia speciosa

Ascorbic Acid 3.35 India In vitro evaluation of free radical scavenging activities of

Panax ginseng and lagertoemia speciosa

Basilici herba 57.15 India Assesment of antioxidant activity of Basili herba

aqueous extract- in vitro studies

Syzygium jambos* 37.65 Puerto Rico Scavenging and antioxidant activity of plant extracts

towards superoxide radical anion R

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Relevance of this Research

Pathophysiological Importance

– “Diabetes and obesity are also

associated with the

overproduction of

mitochondrial reactive oxygen

species (Superoxide), leading

to mitochondrial and cellular

oxidative damage.

– This, in turn, contributes to the

development and progression

of diabetic complications and

to worsening of the diabetic

state per se.” William Sivitz,

MD, Endocrinologist

Etnopharmacological Importance

• To study plants with antioxidant and

free radicals scavenging activity has

relevance in the screening of

possible therapeutics for

hyperglycemia and diabetes.

• Anti-diabetic activity of medicinal

plants and its relationship with

their antioxidant property, India,

2003

– Triphala – A mix of thre plants

T.chebula, T.belerica, E. Officinalis.

http://www.free-press-release.com/news/200803/1206625212.html

1. Literature Review

Read Scientific Papers of Researches related to your area of interest.

2. Do a Protocol of your Research How you are going to do your Research?

Use the literature review as background.

Follow the Scientific Method.

Concentrations Inhibition percentage

Scavenging and Antioxidant Activity Towards

Superoxide Radical of Plant Extracts used

as Diabetes Adjuvants

Materials

Methods

3. Follow the methods

established in the protocols.

From Protocol to Research: A Description

From Protocol to Research: A Description

3. Follow the methods

established in the protocols.

4. Data collection & Data analysis

5. Conclusions

6. Publication

Errors in Data! Does not work!

Review protocols and

literature to answer:

What happened? Why it does not work?

Methodology: in vitro Evaluation of Plant Extracts

1 2 3 4 5 6 7 8 9 10 11 12

A B C D E F H I

Scheme 2-A. The in vitro assay recreates a superoxide radical anions environment.

A B C D E

NBT PMS NADH

85uL 25uL 5uL 25uL

Test

Blanc of the Test

NADH Blanc

Research Progress Step by Step

Literature Review

Making Protocols

Data Collection

January 2010 May 2010 January 2011 May 2011 Aug 2011 Oct 2011

Conclusions Summary

Diabetes Adjuvants Aqueous Extracts Methanolic Extracts

Sizigium jambos 37.65±15.51 79.24±23.85

Tapeinochilus ananassae 188.79±66.21 1032.29±265.25

Costus speciosus 822.02±93.51 1889.29±110.28

Tradescantia spathacea 1,132.4±275.77 3,870.4±353.88

Diabetes Adjuvants

Around the world, mostly in India,

several studies have been conducted

to discover good antioxidant and

scavenging plants extracts, in order to

develop new therapeutics to treat

diabetes.

Six plants extracts were found in the

databases of investigations around the

world, showing superoxide radical

scavenging activity.

In our case, S.jambos aqueous and

methanolic extract, showed IC50’s of

37.65g/mL and 79.24g/mL

respectively.

Our results suggest that S.jambos

is a good antioxidant and

therefore, more studies to screen

phytochemicals that scavenge

superoxide radicals is

recommended to develop new

therapeutic methods to treat

diabetes.

IC50 of Diabetes Adjuvants and

superoxide scavengers Adjuvants around the world IC50’s

Spondia spinatta 13.46μg/ml

Basilici herba 57.15g/mL

Panax ginseng 3.18μg/mL

Lagerstroemia speciosa 6.15μg/mL

Ervatamia coronaria 118.8μg/mL

Referencias

1. 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

2. Francesco Cosentino, MD PhD., High Glucose Increases Nitric Oxide Synthase Expression and Superoxide

Anion Generation in Human Aortic Endothelial Cells, 2002

3. William Sivitz, MD, Mitochondrial Dysfunction in Obesity and Diabetes, US Endocrinology, 2010;6:20-7

4. Kuniharu Suzumura, Superoxide Anion Scavenging Properties of Fluvastatin and Its Metabolies, 1999

5. C. Hu, Dandelion (Taraxacum officinale) flower extract suppresses both reactive oxygen species and nitric oxide

and prevents lipid oxidation in vitro, 2003

6. Nripendranath Mandal, Antioxidant and free radical scavenging activity of Spondias pinnata, 2008

7. Gavillán-Suárez, J., Alvarado-Guzmán, J. & Germosén-Robineau, L. (2009). TRAMIL ethno-pharmacological

survey: Knowledge distribution of medicine plant use in the southeast region of Puerto Rico. Puerto Rico Health

Sciences Journal, 28(4), 329-339.

8. K Suzumura, M Yasuhara, H Narita, Superoxide anion scavenging properties of fluvastatin and its metabolites.

Chem Pharm Bull (Tokyo) 1999 October

Research Skills Developed

Software Applications for Statistical Analysis

SPSS •Graphic Analysis •Linear and logarithmic Regressions

Excel (Microsoft 2007 and For Macintosh 2011)

•Graphic Analysis •Linear and logarithmic Regressions •Standard Deviations •Error Bars •Error Percentage •Platforms using Java

Microplate Reader

pH Meter

Sonicateur Apparatus

Micropipets

Research Skills Developed Laboratory Equipment used in this Research

Interdisciplinary Work

Through all these processes the “Phytochemical and Pharmacognocial lab taught me:

-Have patience. -Learn English as a second language

-Writing skills (Essays, Abstracts, introductions, summaries) - Do Scientific papers and a poster! -M.D. or Ph.D. as final career goals.

-To have more confidence on me. -Share our things. We are a family! -Be responsible in our stuff. -Work hard and you will gain success. -Never gave up! -The capacity to hear and act.

This research experience with Dr. Jannette Gavillán and the Interdisciplinary Research Institute started in October 2009 and brought me several opportunities:

Interdisciplinary Work

October 2009 October 2010 October 2011

October 2009 Started in the PPRL and III

January 2010 Started the Superoxide

and Diabetes Research

Summer 2010 I went to

North Carolina Research

Experience

January 2011 NASA

Scholarship

August 2011 PR-LSAMP Fellowship

November 2011

ABRCMS Travel Award

Interdisciplinary Work

January 2012

Future Goals in Research

May 2012

March 10 UPR Carolina

PR-LSAMP 32th PR

Interdisciplinary Meeting

Post-Bac in University of

Missouri-Columbia

August 2012 August 2013

Graduate School “Free Radical and

Radiation Biology”

This research experience with Dr. Jannette Gavillán and Interdisciplinary Research Institute started in October 2009 and brought me several opportunities:

Interdisciplinary Work

AMP 6th Transdisciplinary Research Conference

NC State Summer Research

Opportunity

Annual Biomedical Research Conference for Minority Students

Summer 2010 May 2011 November 2011

Thank you for your attention!