Gradient solvent fractionation of Costus spiralis methanolic extract

7/30/2019 Gradient solvent fractionation of Costus spiralis methanolic extract

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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



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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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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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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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vinilic Hs


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Hs C

heteroatoms


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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




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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

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Gradient solvent fractionation of Costus spiralis methanolic extract