Quantitative analysis of phytochemicals in antidiabetic plant extracts

Natalie Rivera Ortiz Mentor: Dr. Jannette Gavilln Surez, Ph.D Department of Chemistry CHEM -4999

Acknowledgements
Thanks to: Dr. Jannette Gavilln Surez, 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
Isoprene Ursolic Acid

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 g/mL) stimulated an increase of 228% in insulin release from rat pancreatic -cells compared to the control (5.6 mM glucose).7

Goal and Objectives

Goal: Quantify possible biomarkers of antidiabetic activity of plant extracts from several genera. Objectives: Quantify flavonoids, total phenolic compounds and saponins in plant extracts from Tapeinochuilus annanassae, Syzygium jambos, Costus speciosus, and Tradescantia spathacea.

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 L of AlCl3 10%and 200 L 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 L of Lowry reactive C and 200 L of Lowry reactive E

Quercetin Calibration Curve

Measure absorbance at 760 nm and determine concentration.

Results - Flavonoids
0.8000 0.7000

Calibration curve for determination of flavonoids in antidiabetic plant extracts

Absorbance (415 nm)

0.6000 0.5000 0.4000 0.3000 0.2000 0.1000 0.0000 0.00E+00

y = 34.8x - 0.055 R = 0.994

5.00E-03

1.00E-02

1.50E-02

2.00E-02

2.50E-02

[Quercetin] (mg/mL)

Results - Flavonoids
Calculations
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

Results - Flavonoids
Calculations
Flavonoid content by dry weight: (0.0224 mg/mL QE)(1mL) = 29.8 mg QE/g DW 0.000819 g DW

Results - Flavonoids
Flavonoid content in antidiabetic plants
T. spathacea

Plant

Concentration of flavonoids ( SD mg QE/ g DW) 13.7 0.7 15.8 0.9 24.3 1.3 29.8 0.0

T. spathacea
C. speciosus Plant

C. speciosus
T. anannassae

S. jambos
S. jambos 0 5 10 15 20 25 [Flavonoid] (mgQE/g DW) 30 35

T. anannassae

Results Total Phenols

Calibration curve for determination of total phenolic content 1.0000
0.9000

Absorbance (760 nm)

0.8000 0.7000

0.6000
0.5000 0.4000 0.3000

y = 4.63x + 0.16 R = 0.990

0.2000
0.1000 0.0000 0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

Quercetin concentration (mg/mL)

Results Total Phenols

Calculations
Concentration of methanolic extract in assay: C1V1=C2V2 C2 = C1V1/V2
C2 = (0.150L)(3.00 mg/mL) (1500 L) 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

Results Total Phenols

Calculations
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)

Results Total Phenols

Aqueous extract Total Phenolic Content ( SD mg QE) 0.26 0.01 0.35 0.01 0.45 0.09 Methanolic extract Total Phenolic Content ( SD mg QE) 0.052 0.002 0.060 0.002 0.18 0.04

T. spathacea C. speciosus S. jambos

T. spathacea C. speciosus S. jambos

T. anannassae 0.14 0.02

T. anannassae 0.034 0.005

Results Total Phenols

Total Phenolic Content in Aqueous and Methanolic Extracts
T. spathacea C. speciosus T. anannassae S. jambos 0 5 Content (mg QE/mg 10 extract) Total Phenolic 15 aqueous methanolic

Plant

Current Work
Concentrated saponins are extracted twice with equal volumes of n-butanol

DNS Method
Quantitative determination of glucose. Glucose (0.4%) and 500 L of DNS solution.

Calibration curve of Dglucose (540 nm)

Reaction:

3,5-dinitrosalicylic D-glucose 3-amino-5D-gluconic acid nitrosalicylic acid acid

Current Work
D-glucose calibration curve for determination of saponins
1.4000

1.2000

Absorbance

1.0000

0.8000

0.6000

y = 7.390x - 0.003 R = 1.000

0.4000

0.2000

0.0000 0.000

0.020

0.040

0.060

Concentration (mg/mL)

0.080

0.100

0.120

0.140

0.160

0.180

Current Work
Sugar content in Quijalla saponin is determined from difference between hydrolyzed and non-hydrolyzed saponins
[Glucose] (mg/mL)

Sugar content in Quillaja saponin solutions

[Quillaja saponin] (mg/mL)

Quillaja saponin

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. Rodrguez-Tirado, K.; Gavilln-Surez. 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 Biological Significance. Int.J.Mol.Sci. [Online] 2008, 8, 950-988. http://www.mdpi.org// (accessed March 8, 2011). 3. Gl-nstnda, .; Mazza, G. Saponins: properties, applications 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.; Arajo, 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-induceddiabetic 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 Gavilln Surez, Ph.D Department of Chemistry CHEM -4999