INHIBITION OF -AMYLASE BY PLANT EXTRACTS USED AS DIABETES ADJUVANTS IN PUERTO RICO

Evelyn I. Ortiz Alicea Department of Chemistry Mentor: Jannette Gavilln-Surez, Ph.D.

AKNOWLEDGMENT

Dr. Jannette Gavilln-Surez

WHAT MOTIVATED THIS STUDY?

DIABETES IN OUR SOCIETY

Prevalence of diabetes in Puerto Rico, 2001-2007 In the past seven years the prevalence of diabetes has risen from 9.8% in 2001 to 12.5% in 2007, except for 2004 and 2006 in which decreased to 10.7% and 11.9% respectively.

WHAT IS DIABETES MELLITUS?

Diabetes mellitus (DM) is a chronic metabolic disorder characterized by the presence of high levels of sugar (glucose) in blood, also known as hyperglycemia. 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

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 to decrease the post-prandial hyperglycemia. This is done by retarding the absorption of glucose through the inhibition of the carbohydrate-hydrolyzing enzymes -glucosidase and -amylase in the digestive tract.

Funke, I. et al, 2008

Diverse therapeutic strategies for the treatment of Type 2 are known. The aim of this study was the screening 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.

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 extracts with their -amylase inhibitory activity.

RELEVANCE OF THIS RESEARCH

To our knowledge, these are the first studies of the -amylase inhibitory activity using these extracts at different concentrations to determine IC50.

METHODOLOGY

INHIBITION OF -AMYLASE
-Amylase + starch maltose + DNS reagent A540nm

MALTOSE CALIBRATION CURVE

Solution Maltose Solution 1.8% Deionized Water Blank (L) 0.0 0.01% (L) 5.0 0.02% (L) 10.0 0.03% (L) 15.0 0.04% (L) 20.0 0.05% (L) 25.0

900.0

895.0

890.0

885.0

880.0

875.0

DNS Heat

100.0 15min. a 85C

100.0 15min. a 85C

100.0 15min. a 85C

100.0 15min. a 85C

100.0 15min. a 85C

100.0 15min. a 85C

We prepared a maltose solution of 1.8% (w/v)

EXPECTED RESULTS

INHIBITORS OF -AMYLASE
-Amylase + starch + inhibitor maltose + DNS reagent Quercetin Acarbose Syzygium Jambos

-Amylase Inhibition Bioassay

Ci 8U/mL --0.5% w/v -----

Cf 1U/mL --0.25%w/v -----

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Control 100L amilasa 100L DMSO 400L de almidn 100L H2O 100L DNS Incubar 3 min a temperatura ambiente 15 min a 85C 900L H2O

Blanco 100L Control amilasa 100L DMSO 400L de almidn 100L DNS 100L H2O Incubar 3 min a temperatura ambiente 15 min a 85C 900L H2O

Quercetina 100L amilasa Quercetina DMSO 400L de almidn 100L H2O 100L DNS Incubar 3 min a temperatura ambiente 15 min a 85C 900L H2O

Blanco 100L amilasa Quercetina DMSO 400L de almidn 100L DNS 100L H2O Incubar 3 min a temperatura ambiente 15 min a 85C 900L H2O

Incubar 5 minutos a 37C [ ] g/mL 400 Cantidad 100 de Quercetina (L) Ci=3,200g/m L Cantidad de DMSO (L) 0

300 75

250 62.5

200 50

150 37.5

25

37.5

50

62.5

EXPECTED RESULTS
In order to obtain relevant information it is necessary to linearize the sigmoid curves, which is achieved by expressing the dose in the x-axis logarithmically. From this linear dose-response curve we can calculate the DL50 and the slope of the line, which are the two parameters that can be used to compare the toxicity of two different substances.

Ci 8U/mL --0.5% w/v -----

Cf 1U/mL --0.25%w/v -----

Control Control Blank Acarbose Blank 100L de - 100L de - 100L de - 100L de amilasa amilasa amilasa amilasa 100L 100L Acarbose Acarbose DMSO DMSO DMSO DMSO 400L starch 400L starch 400L starch 400L starch 100L H2O 100L DNS Incubate 3 min @ room temperature 100L H2O 100L DNS Incubate 3 min @ room temperature 100L H2O 100L DNS Incubate 3 min @ room temperature 100L H2O 100L DNS Incubate 3 min @ room temperature

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15 min @ 85C 900L H2O 300 30

15 min @ 85C 900L H2O 250 25

15 min @ 85C 900L H2O 200 20

15 min @ 85C 900L H2O 150 15

Incubate 5 minutes @ 37C [ ] g/mL 400 40 Acarbose volume (L) Ci=3,200g/m L DMSO volume (L)

10

15

20

25

Ci --0.5% w/v --4U/Ml ---

Cf --0.25%w/v --1U/mL ---

Control 40L DMSO 400L de almidn 160L H2O 200L de amilasa Incubar 3 min a temperatura ambiente 100L DNS

Blanco 40L DMSO Control 400L de almidn 160L H2O 100L DNS 200L de amilasa

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Incubar 3 Incubar 3 min a min a temperatura temperatura ambiente ambiente 15 min a 15 min a 15 min a 15 min a 85C y 85C y 85C y 85C y enfriar en enfriar en enfriar en enfriar en bao de hielo bao de hielo bao de hielo bao de hielo 900L H2O 900L H2O 900L H2O 900L H2O

Acarbosa Acarbosa DMSO 400L de almidn 160L H2O 200L de amilasa Incubar 3 min a temperatura ambiente 100L DNS

Blanco Acarbosa DMSO 400L de almidn 160L H2O 100L DNS 200L de amilasa

Incubar 5 minutos a 37C [ ] g/mL 400 Cantidad 40 de Acarbosa(L) Ci=0.1mM Cantidad de DMSO (L) 0

300 30

250 25

200 20

150 15

10

15

20

25

EXPERIMENTAL RESULTS

RESULTS FOR THE MALTOSE CALIBRATION CURVE

The calibration curve always 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 we changed
Maltose 1.8% (w/v) (w/v) Maltose 0.45%

RESULTS FOR THE BIOASSAY OF ACARBOSE

Equation obtain from the graph: y = 0.244x + 38.624 IC50= 46.62 g/mL

Results for the bioassay of Syszygium jambos (methanolic)

Equation obtain from the graph: y = -0.1447x + 30.133 IC50= -137.30 g/mL

NEXT STEP
Now that we have complete the methodology for this study we plan to complete the aims proposed at the beginning of this research. Which were:
Determine the in vitro inhibitory activity (IC50) on porcine 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.

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-phytotherapeutics as inhibitors of -amylase activity. Braz. J. Phramacogn. [Online], 2006. Proquest Direct Web. http://proquest.umi.com/login (access February 7, 2008). Truestar Health Encyclopedia Home Page. http://www.truestarhealth.com (accessed April 21, 2008), Amylase Inhibitors Note. Ali, H.; Houghton, P.; Soumyanath, A.; -Amylase inhibitory activity of some Malaysian plants 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 (accessed February 16, 2008). McCue, P.; Shetty, K.; Inhibitory effects of rosmarinic acid extracts on porcine pancreatic 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 Amaranthus caudatus Seeds. Biol. Pharm. Bull [Online], 2005. Proquest Direct Web. http://proquest.umi.com/login (access May 4, 2008).

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 Cell Phisiol. [Online], 1965. Science Direct. www.sciencedirect.com (access November 1, 2008.) Conforti, F.; Loizzo, M.; Statti, G.; Menichini, F.; Comparative Radical Scavenging and Antidiabetic 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 Biological Materials. 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) y Adiantum poiretti Wikstr (Culantrillo). [Online] (Access June 19, 2008). Chethan, S.; Sreerama, Y.; Malleshi, N.; Mode of inhibition of finger millet malt amylase by the 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 traditional medicine against angiotensin converting enzyme (ACE) and digestive enzymes related to diabetes. 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.).

THANK YOU!!