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Frances Y. Rivera Nieves Jannette Gavilln, PhD University of Puerto Rico at Cayey Chemistry Department QUIM 4999
Diabetes 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
Flavonoids
Antioxidant polyphenolic compounds found in vascular plants, particularly in the flowers, 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 an important role in the protection against oxidative damage and have therapeutic effects over a great number of pathologies, including diabetes mellitus.
Specific Aims
To fractionate Costus spiralis methanolic extracts through a gradient solvent column chromatography 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.
Costus spiralis
Costus spiralis is a ginger, widely use in puertorican folk medicine to treat diabetes mellitus.
Costus sp.
Costus sp.
Flavonols in leaves
Kaempferol 4-methyl ether (Kaempferide) 3-Neohesperidoside Quercetin 4-methyl ether (Tamarixetin) 3-Neohesperidoside
Flavonols in leaves
3,5-dihydroxy-7,4-dimethoxyflavone 3-O-neohesperidoside
Experimental Design
Use thin-layer chromatography (TLC) to identify polarities and antioxidant characteristics of Costus sp. extracts components. Develop a gradient solvent column chromatography to fractionate Costus sp. extracts according to polarity differences. Analyze Costus sp. fractions by Nuclear Magnetic Resonance (1H-NMR) to obtain preliminary spectroscopy data of biomarkers.
TLC
Thin-layer plate
Silica gel
2,2-di(4-tert-octylphenyl)-1-pieryl-hydrazyl (DPPH)
Column Chromatography
Author
Ygartua (1978)
Column conditions
-10-15g of methanolic extract -8 L CH2Cl2:MeOH (85:15) until negative Liebermann reactive reaction -2 L MeOH:H2O (50:50)
Observations
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 -25g ethyl acetate extract -Silica gel -CH2Cl2:MeOH (10:1), (5:1), (2:1) -12 fractions -Sephadex Flavonols in garlic leaves and shoots
Zhao (2007)
Column Chromatography
Author
Awaad (2006)
Column conditions
-30g ethyl acetate extract -Silica CH2Cl2:MeOH (99:1) -Fractions subject to Silica EtoAc:MeOH (gradient) -Preparative TLC -Sephadex with MeOH:H2O -Silica Hexane:EtoAc y MeOH (gradient) -Preparative HPLC -50g ethyl acetate extract -Silica CH2Cl2:MeOH (gradient) -Extract in CH3CN -Amberlite CH3CN (8-50%)/ H2O -HPLC
Observations
Phenolic antioxidants
Column Chromatography
CH2Cl2:MeOH
Column specifications
Column Chromatography
Costus sp. methanolic extract
CH2Cl2:MeOH gradient
Fractions recollection
Rf = 0.76 Rf = 0.72
Rf = 0.74 Rf = 0.62
Rf = 0.52
Rf = 0.53
Rf = 0.55
Rf = 0.41 Rf = 0.40 Rf = 0.25 Rf = 0.22 Rf = 0.16 Rf = 0.10 Rf = 0.05 Rf = 0.07 Rf = 0.03 Rf = 0.21 Rf = 0.20 Rf = 0.19
Rf = 0.06
Frac. 17
Frac. 18
Frac. 19
Frac. 20 Frac. 21
Frac. 10
Frac. 11 Frac. 12
Frac. 13
Frac. 14
Frac. 15
Quercetin
Frac. 1-2
Frac. 16
Frac. 22
Frac. 3
Frac. 4
Frac. 5
Frac. 6 Frac. 7
Frac. 8
Frac. 9
Rutin
BIOMARKERS
Quercetin
Li, 2004
1H-NMR:
BIOMARKERS
Triterpenes
-Amyrin
1H-NMR
Fractions 1-2
aromatic Hs
Hs C heteroatoms
aliphatic Hs
Fractions 3-4
Fractions 5-6
vinilic Hs
Fractions 11-13
Hs C heteroatoms
Fractions 14-19
CONCLUSIONS
The solvent gradient used in column chromatography didnt achieve the expected separation of Costus sp. extract.
1H-NMR
spectra suggests the presence of compounds like terpenes more than flavonoids.
Future Works
Improve column chromatography conditions. 1H-NMR analysis of flavonoids and terpenes standards. Repeat column chromatography to increase fractions quantity and submit them to antidiabetic activity bioassays.
Acknowledgments
Dra. Jannette Gavilln Carmen Cordero, Research Assistant Dra. Claudia Ospina NIH-RIMI Program #1-P2MD001112-0 Instituto de Investigaciones Interdisciplinarias
Chromatography Results
Poor separation in fractions 7-10. Low polarity of fractions 20-22 may suggest that diffusion occurred through the silica. Combination of fractions
Oxidative stress
ANTIOXIDANTS
FREE RADICALS
Susceptibility to infection
Cardiovascular disease
Glucose auto-oxidation
Oxidative stress
Tissue damage
Nephrophaty
Retinophaty