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Gradient solvent fractionation of Costus spiralis methanolic extract

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


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.

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

Costus 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

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.


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)

Column Chromatography
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)


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
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


Phenolic antioxidants

Kumazawa (2007) He (2006) Norbaek (2000)

Ex. Quercetin: Silica CH2Cl2:MeOH (50:1)

Column Chromatography

Solvent (mobile phase) gradient


(100:0) (75:25) (50:50) (0:100)

Adsorbent (stationary phase)

Silica gel Diameter: 2.0 cm Height: 16.0 cm

Column specifications

Column Chromatography
Costus sp. methanolic extract

Silica gel column

CH2Cl2:MeOH gradient

Fractions recollection

TLC and 1H-NMR analysis


Rf = 0.86 Rf = 0.78 Rf = 0.78 Rf = 0.76 Rf = 0.77 Rf = 0.74

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

Catechin Costus sp.


Frac. 1-2

Frac. 16

Frac. 22

Frac. 3

Frac. 4

Frac. 5

Frac. 6 Frac. 7

Frac. 8

Frac. 9



Li, 2004






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


The solvent gradient used in column chromatography didnt achieve the expected separation of Costus sp. extract.

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.


Dra. Jannette Gavilln Carmen Cordero, Research Assistant Dra. Claudia Ospina NIH-RIMI Program #1-P2MD001112-0 Instituto de Investigaciones Interdisciplinarias

Gradient solvent fractionation of Costus spiralis methanolic extract

Frances Y. Rivera Nieves Jannette Gavilln, PhD University of Puerto Rico at Cayey Chemistry Department QUIM 4999

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

1-2 3-4 5-6 7-8 9-10 11-13 14-19 20-22

Oxidative stress


Abnormal AOX levels

Susceptibility to infection

Cardiovascular disease

Glucose auto-oxidation

Oxidative stress

Tissue damage


Glycosylated proteins excess Neurophaty