Quantitative analysis of

phytochemicals in antidiabetic

plant extracts

Natalie Rivera Ortiz

Mentor: Dr. Jannette Gavillán Suárez, Ph.D

Department of Chemistry

CHEM -4999

Acknowledgements

Thanks to:

Dr. Jannette Gavillán Suárez, Ph.D.

What are phytochemicals?

They are secondary metabolites that occur

naturally in plants.

Molecules responsible for the color and organoleptic

properties.

They are considered beneficial in treating and/or

preventing chronic diseases.

http://www.tjclark.com/phytochemicals//

Classification of Phytochemicals

Alkaloids

Nicotine

Cardiac glycosides

Oleandrin

Classification of Phytochemicals

Saponins

Solanine

Terpenoids

Ursolic Acid Isoprene

Classification of Phytochemicals

Phenols

Flavonoids

Quercetin

Previous Studies

Qualitative experiments have demonstrated the

presence of flavonoids, terpenoids, saponins, tannins

and cardiac glycosides in our plant extracts.1

Phenolic compounds protect against oxidative

stress and degenerative diseases.2

Oleanolic acid (saponin) has been reported to have

hypoglycemic activity.3

In vivo antidiabetic activity of several plant extracts

has been correlated with their total phenol and

flavonoid content.4, 5

Previous Studies

Alkaloids, glycosides, carbohydrates, and steroids

have demonstrated activity consistent with their

possible use in treatment of type-2 diabetes.6

Terpenoids have been shown to decrease blood

glucose levels in animal studies.6

Aqueous extract of Acacia arabica (100 μg/mL)

stimulated an increase of 228% in insulin release from

rat pancreatic β-cells compared to the control (5.6 mM

glucose).7

Goal and Objectives

Objectives:

Quantify flavonoids, total phenolic compounds

and saponins in plant extracts from Tapeinochuilus

annanassae, Syzygium jambos, Costus speciosus, and

Tradescantia spathacea.

Goal:

Quantify possible biomarkers of antidiabetic

activity of plant extracts from several genera.

Relevance of this Research

Quantification of phytochemicals will be key

in identifying biomarkers of antidiabetic activity

of these plants.

For the first time, biological activities will be

correlated with phytochemicals’ concentrations.

Methodology

Flavonoids Quercetin

solution(0.27

mg/mL)/plant

extracts , 200

μL of AlCl3

10%and 200 μL

of CH3COONa

1M

Quercetin

Calibration

Curve

Measure

absorbance at

415 nm and

determine

concentration

Methodology

Total Phenolic Content

Quercetin

solution (3.38

mg/mL )/plant

extracts, 600 μL

of Lowry

reactive C and

200 μL of Lowry

reactive E

Quercetin

Calibration

Curve

Measure

absorbance at

760 nm and

determine

concentration.

Results - Flavonoids

y = 34.8x - 0.055 R² = 0.994

0.0000

0.1000

0.2000

0.3000

0.4000

0.5000

0.6000

0.7000

0.8000

0.00E+00 5.00E-03 1.00E-02 1.50E-02 2.00E-02 2.50E-02

Ab

sorb

an

ce (

415

nm

)

[Quercetin] (mg/mL)

Calibration curve for determination of flavonoids in antidiabetic plant extracts

Results - Flavonoids

Concentration of plant

extract in assay:

C1V1=C2V2

C2 = C1V1/V2

C2 = (0.100mL)(0.04094 g/mL)

(5.0000 mL)

C2 = 0.000819 g/mL DW

Flavonoid content in plant

extract:

y = mx+b

x = y-b/m

x = 0.7832-0.002

31.9

x = 0.0244 mg/mL QE

Calculations

Calculations

Results - Flavonoids

Flavonoid content by dry weight:

(0.0224 mg/mL QE)(1mL) = 29.8 mg QE/g DW

Flavonoid content by dry weight:

(0.0224 mg/mL QE)(1mL) = 29.8 mg QE/g DW

0.000819 g DW

Results - Flavonoids

Plant Concentration

of flavonoids

(± SD mg

QE/ g DW)

T. spathacea 13.7 ± 0.7

C. speciosus 15.8 ± 0.9

S. jambos 24.3 ± 1.3

T. anannassae 29.8 ± 0.0 0 5 10 15 20 25 30 35

S. jambos

T. anannassae

C. speciosus

T. spathacea

[Flavonoid] (mgQE/g DW)

Pla

nt

Flavonoid content in antidiabetic plants

Results – Total Phenols

y = 4.63x + 0.16 R² = 0.990

0.0000

0.1000

0.2000

0.3000

0.4000

0.5000

0.6000

0.7000

0.8000

0.9000

1.0000

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18

Ab

sorb

an

ce (

760 n

m)

Quercetin concentration (mg/mL)

Calibration curve for determination of total phenolic content

Results – Total Phenols

Concentration of

methanolic extract in

assay:

C1V1=C2V2

C2 = C1V1/V2

C2 = (0.150μL)(3.00 mg/mL)

(1500 μL)

C2 = 0.300 mg/mL extract

Total phenolic content in

methanolic extract:

y = mx+b

x = y-b/m

x = 0.5603-0.16

4.63

x = 0.085 mg/mL QE

Calculations

Results – Total Phenols

Flavonoid content in methanolic extract:

(0.085 mg/mL QE)(1mL) = 0.29 mg QE/mg extract

0.300 mg extract

Flavonoid content in plant:

(0.29 mg QE)(1 mg extract)(1.19 g extract) = 95.33 mg QE/g DW

(1 mg extract)(1x10-3g extract)(3.62 g DW)

(95.33mg QE)(3.62 g DW) = 7.6 mg QE/ g fresh leaves

(1 g DW)(45.66 g fresh leaves)

Calculations

Results – Total Phenols

Aqueous

extract

Total

Phenolic

Content

(± SD mg

QE)

T. spathacea 0.26 ± 0.01

C. speciosus 0.35 ± 0.01

S. jambos 0.45 ± 0.09

T. anannassae 0.14 ± 0.02

Methanolic

extract

Total

Phenolic

Content

(± SD mg

QE)

T. spathacea 0.052 ± 0.002

C. speciosus 0.060 ± 0.002

S. jambos 0.18 ± 0.04

T. anannassae 0.034 ± 0.005

Results – Total Phenols

0 5 10 15

S. jambos

T.anannassae

C. speciosus

T. spathacea

Total Phenolic Content (mg QE/mg extract)

Pla

nt

Total Phenolic Content in Aqueous and Methanolic Extracts

aqueous

methanolic

Current Work

Concentrated

saponins are

extracted twice

with equal

volumes of

n-butanol

DNS Method Quantitative

determination

of glucose.

Glucose (0.4%)

and 500 μL of

DNS solution.

Calibration

curve of D-

glucose

(540 nm)

3,5-dinitrosalicylic

acid D-glucose 3-amino-5-

nitrosalicylic acid

D-gluconic

acid

Reaction:

Current Work

y = 7.390x - 0.003 R² = 1.000

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

1.4000

0.000 0.020 0.040 0.060 0.080 0.100 0.120 0.140 0.160 0.180

Ab

sorb

an

ce

Concentration (mg/mL)

D-glucose calibration curve for determination of saponins

Current Work

Sugar content in

Quijalla saponin

is determined

from difference

between

hydrolyzed and

non-hydrolyzed

saponins

Quillaja saponin

Sugar content in Quillaja

saponin solutions

[Quillaja saponin] (mg/mL)

[Glu

cose

] (m

g/m

L)

Future Work

Determination of saponin content in plant

extracts from Tapeinochuilus annanassae, Syzygium

jambos, Costus speciosus, and Tradescantia spathacea.

Determination of alkaloid content in plant

extracts.

References

1. Rodríguez-Tirado, K.; Gavillán-Suárez. University of Puerto Rico

at Cayey, Cayey, P.R. Phytochemical studies of medicinal

plants from several genera with antidiabetic properties.

Unpublished work, 2011.

2. Han, X.; Shen, T.; Lou, H. Dietary polyphenols and Their Biolo-

gical Significance. Int.J.Mol.Sci. [Online] 2008, 8, 950-988.

http://www.mdpi.org// (accessed March 8, 2011).

3. Güçlü-Ünstündağ, Ö.; Mazza, G. Saponins: properties, applica-

tions and processing. Crit. Rev. Food Sci. Nutr. [Online] 2007,

47, 231-258. http://www.redorbit.com// (accessed July 14,

2011).

References

4. Rauter, A.P.; Martins, A.; Lopes, R.; Ferreira, J.; Serralheiro, L.M.;

Araújo, M.E.; Borges, C.; Justino, J.; Silva, F.V.; Goulart, M.;

Thomas-Oates, J.; Rodrigues, J.A.; Edwards, E.; Noronha, J.P.;

Pinto, R.; Mota-Filipe, H. Bioactivity studies and chemical

profile of the antidiabetic plant Genista tenera. J.

Ethnopharmacol. [Online] 2009, 122, 384-393. Science Direct.

http://www. sciencedirect.com(accessed March 8, 2011).

5. Aslan, M.; Deliorman Orhan, D.; Orhan, N.; Sezik, E.; Yesilada, E. In

vivo antidiabetic and antioxidant potential of H elichrysum

plicatumssp. plicatum capitulums in streptozotocin-induced-

diabetic rats. J. Ethnopharmacol. [Online] 2007, 109, 54-59.

Science Direct. http://www. sciencedirect.com(accessed

November 20, 2011).

References

6. Kumar, A.; Ilavarasan, R.; Jayachandran, T.; Decaraman, N.;

Aravindhan, P.; Padmanabhan, N.; Krishnan, M.R.V.

Phytochemicals Investigation on a Tropical Plant, Syzygium

cumini fromKattuppalayam, Erode District, Tamil Nadu, South

India. Pak. J. Nutr. [Online] 2009, 8, 1, 83-85.

http://www.pjbs.org/pjnonline// (accessed March 8, 2011).

7. Kaur, L.; Han, K.S.; Bains, K.; Singh, H. Indian culinary plants

enhance glucose-induced insulin secretion and glucose

consumption in INS-1 b-cells and 3T3-L1 adipocytes. Food

Chem. [Online] 2011, 29, 1120-1125. Science Direct.

http://www. sciencedirect.com(accessed November 20,

2011).

Thanks for your attention!

Quantitative analysis of

phytochemicals in antidiabetic

plant extracts

Natalie Rivera Ortiz

Mentor: Dr. Jannette Gavillán Suárez, Ph.D

Department of Chemistry

CHEM -4999