gradient solvent fractionation of costus spiralis methanolic extract
TRANSCRIPT
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Gradient solvent fractionation ofGradient solvent fractionation of
CostusCostus spiralisspiralis methanolicmethanolic
extractextract
Frances Y. Rivera Nievesrances Y. Rivera NievesJannette Gavillannette Gavilln, PhD, PhDUniversity of Puerto Rico at Cayeyniversity of Puerto Rico at CayeyChemistry Departmenthemistry DepartmentQUIM 4999UIM 4999
Frances Y. Rivera Nievesrances Y. Rivera NievesJannette Gavillannette Gavilln, PhD, PhDUniversity of Puerto Rico at Cayeyniversity of Puerto Rico at CayeyChemistry Departmenthemistry DepartmentQUIM 4999UIM 4999
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Diabetes mellitusDiabetes mellitus
In Puerto Rico, 10.6% of adults had diabetes
in 2004, versus 6.7% in USA 7.8% of the United States population (2007)
High blood sugar levels (hyperglycemia) Patients have reduced antioxidant (AOX)
defenses Increase the reactive oxygen species (ROS) levels
Increase the risk of free radical-mediated diseases
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FlavonoidsFlavonoids
Antioxidant polyphenoliccompounds found in vascularplants, particularly in theflowers, leaves and bark.
They are abundant in fruits,vegetables and in beverages
consisting of plant extracts(tea, wine, beer).
Due to their antioxidant
capacity, flavonoids play animportant role in the protectionagainst oxidative damage andhave therapeutic effects over a
great number of pathologies,including diabetes mellitus.
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Specific AimsSpecific Aims
To fractionate Costus spiralis methanolic
extracts through a gradient solvent columnchromatography and perform in vitro
bioassays for anti-diabetic activities on the
fractions.
To identify biomarkers of anti-diabetic
biological activity in the most active fractions.
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CostusCostusspiralisspiralis
Costus spiralis is a ginger,
widely use in puertorican
folk medicine to treat
diabetes mellitus.
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CostusCostussp.sp.
C. pictus (Jothivel, 2007)
Oral anti-diabetic activity (120 mg/Kg/da) Pentacyclic triterpene -Amyrin
C. speciosus (Mosihuzzaman, 1994; Guerrero,
2002)
Oral hypoglycemic activity (1.5g/24h) Increase glucose absortion
Quercetin glycosides
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CostusCostussp.sp.
C. spicatus (Harborne, 2001)
Flavonols in leaves Kaempferol 4-methyl ether (Kaempferide) 3-Neohesperidoside
Quercetin 4-methyl ether (Tamarixetin) 3-Neohesperidoside
C. spiralis (Antunes, 2000; Harborne, 2001)
Flavonols in leaves Kaempferol 7, 4-dimethyl ether 7-Glucoside
Flavonol diglycoside
3,5-dihydroxy-7,4-dimethoxyflavone 3-O-neohesperidoside
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Experimental DesignExperimental Design
Use thin-layer chromatography (TLC) to identify
polarities and antioxidant characteristics ofCostus sp. extracts components.
Develop a gradient solvent column
chromatography to fractionate Costus sp. extractsaccording to polarity differences.
Analyze Costus sp. fractions by NuclearMagnetic Resonance (1H-NMR) to obtainpreliminary spectroscopy data of biomarkers.
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TLCTLC
Mobile phase selection
n-butanol : acetic acid : water (4:1:5) ethyl acetate : methanol : acetic acid (80:20:1)
Thin-layer plate
Silica gel
2,2-di(4-tert-octylphenyl)-1-pieryl-hydrazyl
(DPPH) Solution used to visualize antioxidant compounds
(violet/yellow)
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Column ChromatographyColumn ChromatographyAuthor Column conditions Observations
Ygartua (1978) -10-15g of methanolic extract
-8 L CH2Cl2:MeOH (85:15) until negative
Liebermann reactive reaction
-2 L MeOH:H2
O (50:50)
Alexandre (2000) -Costus n-Butanol extract
-Amberlite MeOH 100% (glycosides)
-Glycosides filtration with Sephadex
Mi-Yeon (2005) -Ethyl acetate extracts
-Amberlite MeOH:H2O (gradient)
-70% MeOH fraction subject to Sephadex with 60%
MeOH
-Semipreparative HPLC
Flavonols in garlic
leaves and shoots
Zhao (2007) -25g ethyl acetate extract
-Silica gel
-CH2Cl2:MeOH (10:1), (5:1), (2:1)-12 fractions
-Sephadex
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Column ChromatographyColumn Chromatography
Author Column conditions Observations
Awaad (2006) -30g ethyl acetate extract
-Silica CH2Cl2:MeOH (99:1)
-Fractions subject to Silica EtoAc:MeOH
(gradient)
-Preparative TLC
-Sephadex with MeOH:H2O
Phenolic antioxidants
Kumazawa (2007) -Silica Hexane:EtoAc y MeOH (gradient)
-Preparative HPLC
He (2006) -50g ethyl acetate extract
-Silica CH2Cl2:MeOH (gradient)
Ex. Quercetin: SilicaCH2Cl2:MeOH (50:1)
Norbaek (2000) -Extract in CH3CN
-Amberlite CH3CN (8-50%)/ H2O-HPLC
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Column ChromatographyColumn Chromatography
Solvent (mobile phase) gradient
CH2Cl
2:MeOH
(100:0)
(75:25)
(50:50)
(0:100)
Adsorbent (stationary phase)
Silica gel
Column specifications Diameter: 2.0 cm
Height: 16.0 cm
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Column ChromatographyColumn Chromatography
Costus sp. methanolic extract
Silica gel column
Fractions recollection
TLC and 1H-NMR analysis
CH2Cl2:MeOH
gradient
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TLC FRACTIONS ANALYSISTLC FRACTIONS ANALYSIS
Ru
tin
Quercetin
Catech
in
Costussp.
Frac.1
-2
Frac
.3
Frac
.4
Frac
.5
Frac
.6
Frac
.7
Frac
.8
Frac
.9
Frac.
10
Frac.
11
Frac.
12
Frac.
13
Frac.
14
Frac.
15
Frac.
16
Frac.
17
Frac.
18
Frac.
19
Frac.
20
Frac.
21
Frac.
22
Rf= 0.16
Rf= 0.78
Rf= 0.52
Rf= 0.10
Rf= 0.25
Rf= 0.86
Rf= 0.78
Rf= 0.74
Rf= 0.05
Rf= 0.22
Rf= 0.41
Rf= 0.53
Rf= 0.76
Rf= 0.03
Rf= 0.21
Rf= 0.55
Rf= 0.77
Rf= 0.40
Rf= 0.74
Rf= 0.76
Rf= 0.07
Rf= 0.20
Rf= 0.06
Rf= 0.19
Rf= 0.72
Rf= 0.62
Rf= 0.16
Rf= 0.78
Rf= 0.52
Rf= 0.10
Rf= 0.25
Rf= 0.86
Rf= 0.78
Rf= 0.74
Rf= 0.05
Rf= 0.22
Rf= 0.41
Rf= 0.53
Rf= 0.76
Rf= 0.03
Rf= 0.21
Rf= 0.55
Rf= 0.77
Rf= 0.40
Rf= 0.74
Rf= 0.76
Rf= 0.07
Rf= 0.20
Rf= 0.06
Rf= 0.19
Rf= 0.72
Rf= 0.62
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BIOMARKERSBIOMARKERS
Quercetin
Li, 2004
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Triterpenes
-Amyrin
11HH--NMR: BIOMARKERSNMR: BIOMARKERS
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11HH--NMRNMR
Fractions 1-2
aliphatic Hs
Hs C
heteroatomsaromatic Hs
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Fractions 3Fractions 3--44
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Fractions 5Fractions 5--66
vinilic Hs
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Fractions 11Fractions 11--1313
Hs C
heteroatoms
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Fractions 14Fractions 14--1919
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CONCLUSIONSCONCLUSIONS
The solvent gradient used in column
chromatography didnt achieve the expectedseparation ofCostus sp. extract.
1H-NMR spectra suggests the presence of
compounds like terpenes more than flavonoids.
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Future WorksFuture Works
Improve column chromatography conditions.
1H-NMR analysis of flavonoids and terpenesstandards.
Repeat column chromatography to increasefractions quantity and submit them to anti-
diabetic activity bioassays.
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AcknowledgmentsAcknowledgments
Dra. Jannette Gavilln
Carmen Cordero, Research Assistant Dra. Claudia Ospina
NIH-RIMI Program #1-P2MD001112-0 Instituto de Investigaciones Interdisciplinarias
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Gradient solvent fractionation ofGradient solvent fractionation of
CostusCostus spiralisspiralismethanolicmethanolic
extractextract
Frances Y. Rivera Nievesrances Y. Rivera NievesJannette Gavillannette Gavilln, PhD, PhDUniversity of Puerto Rico at Cayeyniversity of Puerto Rico at CayeyChemistry Departmenthemistry DepartmentQUIM 4999UIM 4999
Frances Y. Rivera Nievesrances Y. Rivera NievesJannette Gavillannette Gavilln, PhD, PhDUniversity of Puerto Rico at Cayeyniversity of Puerto Rico at CayeyChemistry Departmenthemistry DepartmentQUIM 4999UIM 4999
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Chromatography ResultsChromatography Results Poor separation in fractions 7-10.
Low polarity of fractions 20-22 may suggest that
diffusion occurred through the silica. Combination of fractions
1-2
3-4 5-6
7-8
9-10 11-13
14-19
20-22
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Oxidative stressOxidative stress
Oxidative stress
Abnormal AOX
levels
Glucose
auto-oxidation
Glycosylated
proteins excess
Tissue damage Nephrophaty
Cardiovascular
disease
Retinophaty
Neurophaty
Susceptibility
to infection
FREE
RADICALS
ANTIOXIDANTS