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AKNOWLEDGMENT
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.
METHODOLOGY
INHIBITION OF -AMYLASE
-Amylase + starch maltose + DNS reagent A540nm
900.0
895.0
890.0
885.0
880.0
875.0
DNS Heat
EXPECTED RESULTS
INHIBITORS OF -AMYLASE
-Amylase + starch + inhibitor maltose + DNS reagent Quercetin Acarbose Syzygium Jambos
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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.
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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Incubate 5 minutes @ 37C [ ] g/mL 400 40 Acarbose volume (L) Ci=3,200g/m L DMSO volume (L)
10
15
20
25
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
Equation obtain from the graph: y = 0.244x + 38.624 IC50= 46.62 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.).
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