inhibition of α-amylase by plant extracts used as diabetes adjuvants in puerto rico
TRANSCRIPT
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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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