Inhibition of α-amylase by plant extracts used as Diabetes adjuvants in Puerto Rico

7/30/2019 Inhibition of -amylase by plant extracts used as Diabetes adjuvants in Puerto Rico

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INHIBITION OF -AMYLASE BY

PLANT EXTRACTS USED AS

DIABETES ADJUVANTS IN

PUERTO RICO

Evelyn I. Ortiz Alicea

Department of ChemistryMentor: Jannette Gavilln-Surez, Ph.D.


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AKNOWLEDGMENT

Dr. JannetteGavilln-Surez


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WHAT MOTIVATED THISSTUDY?


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DIABETES IN OUR SOCIETY

Prevalence of

diabetes in PuertoRico, 2001-2007

In the past sevenyears the prevalence

of diabetes has risenfrom 9.8% in 2001 to12.5% in 2007, except

for 2004 and 2006 inwhich decreased to

10.7% and 11.9%respectively.


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WHAT IS DIABETES MELLITUS?

Diabetes mellitus (DM) is a chronic metabolic

disorder characterized by the presence of highlevels of sugar (glucose) in blood, also known ashyperglycemia.

According to the Committee experts of the

American Diabetes Association (ADA) in 1997,different types of DM are classified into 4 groups:

Diabetes Mellitus Type 1

Diabetes Mellitus Type 2 Gestational Diabetes

Other types of Diabetes Mellitus


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PREVIOUS STUDIES

There have been several studies that aim to inhibit

the high glucose levels in blood.

Raj, M. et al, 2008

One therapeutic approach for treating diabetes is todecrease the post-prandial hyperglycemia. This isdone by retarding the absorption of glucose throughthe inhibition of the carbohydrate-hydrolyzingenzymes -glucosidase and -amylase in the digestive

tract.


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Funke, I. et al, 2008

Diverse therapeutic strategies for the treatment ofType 2 are known. The aim of this study was thescreening for -amylase inhibition of traditionally

plants used in anti-diabetic treatment and pure

natural products.

Valsa et al, 1997

Tea polyphenols have been reported to inhibit -amylase and also are key substances for suppressing

post-prandial hyperglycemia.

Gavilln-Surez et al, 2009

Four medicinal plants that are frequently used as

diabetes adjuvants in the Island were identified.


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THE SPECIFIC AIMS OF THE

PROPOSE STUDY ARE

Determine the in vitro inhibitory activity

(IC50) on porcine pancreatic -amylase of

methanolic and aqueous extracts of:

Costus spiralis Tapeinochilus annassae

Rhoeo spathacea

Syszygium jambos

Correlate the phenolic content of the extractswith their -amylase inhibitory activity.


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RELEVANCE OF THISRESEARCH

To our knowledge, these are the first studiesof the -amylase inhibitory activity using

these extracts at different concentrations to

determine IC50.


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METHODOLOGY


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INHIBITION OF-AMYLASE

-Amylase + starch maltose + DNS reagent A540nm


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MALTOSE CALIBRATION CURVE

We prepared a maltose

solution of 1.8% (w/v)

Solution Blank

(L)

0.01%

(L)

0.02%

(L)

0.03%

(L)

0.04%

(L)

0.05%

(L)

Maltose

Solution

1.8%

0.0 5.0 10.0 15.0 20.0 25.0

Deionized

Water

900.0 895.0 890.0 885.0 880.0 875.0

DNS 100.0 100.0 100.0 100.0 100.0 100.0

Heat 15min.

a 85C

15min.

a 85C

15min.

a 85C

15min.

a 85C

15min.

a 85C

15min.

a 85C


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EXPECTED RESULTS


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INHIBITORS OF-AMYLASE

-Amylase + starch + inhibitor maltose + DNS reagent

Quercetin Acarbose Syzygium Jambos


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Ci Cf Control Blanco

Control

Quercetina Blanco

8U/mL 1U/mL 100L -

amilasa

100L -

amilasa

100L -

amilasa

100L -

amilasa

--- --- 100L

DMSO

100L

DMSO

Quercetina Quercetina

DMSO DMSO

0.5% w/v 0.25%w/v 400L de

almidn

400L de

almidn

400L de

almidn

400L de

almidn

--- --- 100L H2O 100L DNS 100L H2O 100L DNS

--- --- 100L DNS 100L H2O 100L DNS 100L H2O

--- --- Incubar 3

min a

temperatura

ambiente

Incubar 3

min a

temperatura

ambiente

Incubar 3

min a

temperatura

ambiente

Incubar 3

min a

temperatura

ambiente--- --- 15 min a85C

15 min a85C

15 min a85C

15 min a85C

--- --- 900L H2O 900L H2O 900L H2O 900L H2O

Incubar 5 minutos a 37C

[ ] g/mL 400 300 250 200 150

Cantidad

de Quercetina

(L)

Ci=3,200g/m

L

100 75 62.5 50 37.5

Cantidad

de DMSO (L)

0 25 37.5 50 62.5

-Amylase Inhibition Bioassay


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EXPECTED RESULTS

In order to obtainrelevant information it is

necessary to linearizethe sigmoid curves,which is achieved byexpressing the dose inthe x-axis logarith-mically. From this linear

dose-response curve wecan calculate the DL50and the slope of the line,which are the twoparameters that can beused to compare the

toxicity of two differentsubstances.


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Ci Cf Control Control Blank Acarbose Blank

8U/mL 1U/mL 100L de -

amilasa

100L de -

amilasa

100L de -

amilasa

100L de -

amilasa

--- --- 100L

DMSO

100L

DMSO

Acarbose Acarbose

DMSO DMSO

0.5% w/v 0.25%w/v 400L starch 400L starch 400L starch 400L starch

--- --- 100L H2O 100L H2O 100L H2O 100L H2O

--- --- 100L DNS 100L DNS 100L DNS 100L DNS

--- --- Incubate 3

min @ room

temperature

Incubate 3

min @ room

temperature

Incubate 3

min @ room

temperature

Incubate 3

min @ room

temperature

--- --- 15 min @

85C

15 min @

85C

15 min @

85C

15 min @

85C

--- --- 900L H2O 900L H2O 900L H2O 900L H2O

Incubate 5 minutes @ 37C

[ ] g/mL 400 300 250 200 150

Acarbose

volume (L)

Ci=3,200g/m

L

40 30 25 20 15

DMSO

volume (L)

0 10 15 20 25


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Ci Cf Control Blanco

Control

Acarbosa Blanco

--- --- 40L DMSO 40L DMSO Acarbosa Acarbosa

DMSO DMSO0.5% w/v 0.25%w/v 400L de

almidn

400L de

almidn

400L de

almidn

400L de

almidn

--- --- 160L H2O 160L H2O 160L H2O 160L H2O

4U/Ml 1U/mL 200L de -

amilasa

100L DNS 200L de -

amilasa

100L DNS

--- --- Incubar 3

min atemperatura

ambiente

200L de -

amilasa

Incubar 3

min atemperatura

ambiente

200L de -

amilasa

--- --- 100L DNS Incubar 3

min a

temperatura

ambiente

100L DNS Incubar 3

min a

temperatura

ambiente--- --- 15 min a

85C yenfriar en

bao de hielo

15 min a

85C yenfriar en

bao de hielo

15 min a

85C yenfriar en

bao de hielo

15 min a

85C yenfriar en

bao de hielo

--- --- 900L H2O 900L H2O 900L H2O 900L H2O

Incubar 5 minutos a 37C

[ ] g/mL 400 300 250 200 150Cantidad

de

Acarbosa(L)

Ci=0.1mM

40 30 25 20 15

Cantidad

de DMSO

(L)

0 10 15 20 25


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EXPERIMENTAL RESULTS


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RESULTS FOR THE MALTOSE

CALIBRATION CURVE

The calibration curvealways show a R of ~0.99

but once we changed our

positive control we had to

changed the concentration of

maltose because they were

too high and we couldnt

measure thge maltose

concentration with precision.

On April 1, 2011 wechanged

Maltose 1.8% (w/v) Maltose 0.45%

(w/v)


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RESULTS FOR THE BIOASSAY OF

ACARBOSE

Equation obtainfrom the graph:

y = 0.244x + 38.624

IC50= 46.62 g/mL


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Results for the bioassay ofSyszygium jambos

(methanolic)

Equation obtain fromthe graph:

y = -0.1447x + 30.133

IC50= -137.30 g/mL


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NEXT STEP

Now that we have complete the methodology for

this study we plan to complete the aims proposedat the beginning of this research. Which were:

Determine the in vitro inhibitory activity (IC50) onporcine pancreatic -amylase of methanolic and aqueous

extracts of: Costus spiralis

Tapeinochilus annassae

Rhoeo spathacea

Correlate the phenolic content of the extracts

with their -amylase inhibitory activity.


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REFERENCES Raj, M.; Jong-Anurakkun, N.; Hong, G.; Kawabata, J. -Glucosidase and -amylase

inhibitory activities of Nepalese medicinal herb Pakhanbhed (Bergenia ciliate, Haw.).Food Chem. [Online], 2008. Science Direct. www.sciencedirect.com (access January 27,2008).

Funke, I.; Melzig, M.; Traditionally used plants in diabetes therapy-phytotherapeuticsas inhibitors of -amylase activity. Braz. J. Phramacogn. [Online], 2006. ProquestDirect Web. http://proquest.umi.com/login (access February 7, 2008).

Truestar Health Encyclopedia Home Page. http://www.truestarhealth.com (accessedApril 21, 2008), Amylase Inhibitors Note.

Ali, H.; Houghton, P.; Soumyanath, A.; -Amylase inhibitory activity of some Malaysianplants used to treat diabetes; with particular reference to Phyllanthus amarus. J. Ethno

Pharmco [Online], 2006. Science Direct. www.sciencedirect.com (access March 27,2008).

Chaplin, M. Enzymes and Enzyme Technology. http://www.lsbu.ac.uk (accessedFebruary 16, 2008).

McCue, P.; Shetty, K.; Inhibitory effects of rosmarinic acid extracts on porcinepancreatic amylase in vitro. Asia Pacific J. Clin. Nutr. [Online], 2004. Proquest Direct

Web. http://proquest.umi.com/login (access Mach 22, 2008). Conforti, F.; Loizzo, M.; Statti, G.; Menichini, F.; Sacchetti, G.; Poli, F.; In vitro

Antioxidant Effect and Inhibition of - Amylase of Two Varieties of Amaranthuscaudatus Seeds. Biol. Pharm. Bull [Online], 2005. Proquest Direct Web.http://proquest.umi.com/login (access May 4, 2008).


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Ogawa, Y.;Imamura, S.; Effect of Plant Extracts and Gibberellin A3 on -Amylase Production

in Embryoless Rice Endosperm in Relation to Growth- Promoting Activity. Plant and CellPhisiol. [Online], 1965. Science Direct. www.sciencedirect.com (access November 1, 2008.)

Conforti, F.; Loizzo, M.; Statti, G.; Menichini, F.; Comparative Radical Scavenging andAntidiabetic Activities of Methanolic Extrat and Fractions from Achillealinguistica All. Biol.Pharm. Bull 28 (9) 1791-1794, 2005.

Fossum, K.; Whitaker, J.; Simple Method for Detecting Amylase Inhibitors in BiologicalMaterials. J. Nutr. [Online], 1974. www.jn.nutrition.org (access June 9, 2008).

Ojeda, R.; Guerrero, O.; Jaramillo, F.; INHIBICION DE LA ACTIVIDAD DE -AMILASA Y- GLUCOSIDASA A PARTIR DE LOS EXTRACTOS TOTALES DE Justicia colorata (Nees)Wassh (Insulina),Artocarpus altilis (Parkinson) Fosberg (Fruto del pan) yAdiantum poirettiWikstr (Culantrillo). [Online] (Access June 19, 2008).

Chethan, S.; Sreerama, Y.; Malleshi, N.; Mode of inhibition of finger millet malt amylase bythe millet phenolics. Food Chem. [Online], 2008. Science Direct. www.sciencedirect.com(access July 2, 2008).

Loizzo, M.; Saab, A.; In vitro inhibitory activities of plants used in Lebanon traditionalmedicine against angiotensin converting enzyme (ACE) and digestive enzymes related todiabetes. J. Ethnopharmco, [Online] 2008. Science Diect. www.sciencedirect.com (access

January 22, 2009.). Guzman, A.; Jatomea, O.; Robles, M.; Characterization of - amylase inhibitor from Palo

Fierro seeds. Plant Physiol. And Bioquem. [Online] 2007. Science Direct.www.sciencedirect.com (access February 11, 2009.).


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Inhibition of α-amylase by plant extracts used as Diabetes adjuvants in Puerto Rico

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