Spectrophotometric determination of saponins in antidiabetic plant extracts

Natalie Rivera Ortiz Mentor: Jannette Gavilln Surez, Ph.D. Department of Chemistry May 2012

Acknowledgements

Dr. Jannette Gavilln, Ph.D. Chemistry Department Laboratory Technicians

Saponins
Plant materials containing saponins have long been used in many parts of the world for their detergent properties.1 Saponins (Latin sapo, soap) are glycosides that are characterized by their property of producing a frothing aqueous solution. Often they occur as complex mixtures with the components differing in the nature of the sugars present, or in the structure of the aglycone (sapogenin).

Saponins
Two kinds of saponins:
Steroidal saponins Pentacyclic triterpenoid saponins

Digitonin

Quillaja saponin

Phytochemicals and Diabetes

Several plant-derived materials have demonstrated activity consistent with their possible use in the treatment of type 2 diabetes.2 Oleanolic acid (sapogenin) has been reported to have hypoglycemic activity.3 In a previous research, Trigonella foenumgraecum (fenugreek) extracts significantly stimulated insulin release from rat pancreatic INS-1 cells, glucose consumption by mouse 3T3L1 adipocytes and limited glucose movement across a dialysis membrane. Some of its active constituents are saponins.2

Phytochemicals and Diabetes

Qualitative experiments have demonstrated the presence of flavonoids, terpenoids, saponins, tannins and cardiac glycosides in our plant extracts.4 In qualitative results, only S. jambos gave a positive result for saponins, but TLC showed them to be present in all plant extracts.

Our plants

Costus speciosus

Tradescantia spathacea

Syzygium jambos

Tapeinochilus anannassae

Goal and Objective

Goal

Quantify saponins in order to identify possible

biomarkers of antidiabetic activity of plant extracts from several genera. Objective To quantify saponins in plant extracts of Tapeinochilus anannassae, Syzygium jambos, Costus speciosus and Tradescantia spathacea.

Relevance of this Research

Quantification of phytochemicals, such as

saponins, will be key in identifying biomarkers

of antidiabetic activity of these plants.

For the first time, biological activities will be

correlated with saponin concentrations.

http://www.psna-online.org//

Methodology
DNS Method for determination of glucose Calibration curve of Quillaja saponin

Preparation of saponin extracts

Reaction:

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

yellow

red

Methodology
Preparation of Saponin Extracts

10 g of dried and ground samples are defatted with hexane in a Soxhlet apparatus

Saponins are extracted twice with 100 mL of aqueous 80% CH3OH

Extracts are centrifuged a 3000 rpm for 10 min

Methodology
Preparation of Saponin Extracts
CH3OH is evaporated under vacuum to remain with aqueous phase Aqueous phase is extracted 3 times with an equal volume of dichloromethane
Concentrated saponins are extracted twice with an equal volume of nbutanol

Dried fractions are dissolved in 10 mL of water and freeze-dried

Methodology
DNS Method
1.4000

D-glucose calibration curve for determination of saponins

Glucose (0.4%) and 500 L of DNS solution. Measure at 540 nm.

1.2000
1.0000 0.8000 0.6000 0.4000 0.2000 0.0000 0.000 0.050 0.100

y = 7.390x - 0.003 R = 1.000

0.150 0.200

Methodology
Calibration curve of hydrolyzed sugars of Quillaja saponin
[Glucose] (mg/mL)

Hydrolysis of saponins (3-7 mg/mL) 5 mL of 6M HCl + 85C pH 6.5-7.2

Sugar content is determined from difference between hydrolyzed and non-hydrolyzed saponins

Sugar content in Quillaja saponin solutions

[Quillaja saponin] (mg/mL)

Results
0.7000 0.6000 0.5000

D-glucose calibration curve

Absorbance

0.4000 0.3000 0.2000 0.1000 0.0000 0.0000

y = 7.08x + 0.024 R = 1.00

0.0200

D-glucose concentration (mg/mL)

0.0400

0.0600

0.0800

0.1000

Results
Sugar content in Quillaja saponin solutions
0.040

Sugar concentration (mg Dglucose/mL)

0.035 0.030
0.025 0.020 0.015 0.010 0.005 0.000 0.000 0.050 0.100 0.150 0.200 0.250

y = 0.156x - 0.001 R = 0.718

Quillaja saponin concentration (mg/mL)

Results

Calculations

Saponin content in plant extract: y = mx+b x = y-b/m x = 0.0756-0.024 = 0.0073 mg D-glucose/mL 7.08 x = 0.0073+0.001 = 0.0517 mg Q.s./mL 0.156

Results
Saponin concentrations in plant extracts
Plant Saponin concentration (mg Quillaja saponin/mL) 0.0517 0.0655 0.1714

C. speciosus S. jambos T. spathacea T. anannassae

Results
Saponin concentrations in antidiabetic plant extracts
T. spathacea

S. jambos

T. anannassae

C. speciosus 0.0000 0.0500 0.1000 0.1500 0.2000 Saponin concentration (mg Quillaja saponin/mL)

Current work
Optimization of hydrolysis conditions and Quillaja saponin calibration curve . Determination of saponin content in T. spathacea extracts.

Conclusions
T. anannassae obtained the highest saponin
content, with 0.1714 mg Quillaja saponin/mL. The Quillaja saponin curve should be optimized in

order to get a better correlation coeficient.

Our results confirmed the presence of saponins in T. anannassae, C. speciosus and S. jambos extracts, which confirms the TLC results obtained in a previous work.

References
1. 2. Trease and Evans. Pharmacognosy. 1996. WB Saunders Company Limited, London, U.K. 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, 11201125. Science Direct. http://www.sciencedirect.com(accessed November 20, 2011). Gl-nstnda, .; Mazza, G. Saponins: pr operties, applica tions and processing. Crit. Rev. Food Sci. Nutr. [Online] 2007, 47, 231-258. http://www.redorbit.com// (accessed July 14, 2011). Rodrguez-Tirado, K.; Gavilln-Surez J. University of Puerto Rico at Cayey, Cayey, P.R. Phytochemical studies of medicinal plants from several genera with antidiabetic properties. Unpublished work, 2011.

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Thanks for your attention!

Spectrophotometric determination of saponins in antidiabetic plant extracts

Natalie Rivera Ortiz Mentor: Jannette Gavilln Surez, Ph.D. Department of Chemistry