spectrophotometric determination of saponins in antidiabetic plant extracts. natalie rivera....

Spectrophotometric determination

of saponins in antidiabetic plant

extracts Natalie Rivera Ortiz

Mentor: Jannette Gavillán Suárez, Ph.D.

Department of Chemistry

May 2012

Acknowledgements

• Dr. Jannette Gavillán, 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:

Quillaja saponin

Pentacyclic triterpenoid saponins Steroidal saponins

Digitonin

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 foenum-graecum (fenugreek) extracts significantly stimulated insulin release from rat pancreatic INS-1 cells, glucose consumption by mouse 3T3-L1 adipocytes and limited glucose movement across a dialysis membrane. Some of its active constituents are saponins.2

• 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.

Phytochemicals and Diabetes

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

Preparation of saponin extracts

DNS Method for determination of

glucose

Calibration curve of Quillaja

saponin

Reaction:

3,5-dinitrosalicylic acid

D-glucose 3-amino-5-nitrosalicylic acid

D-gluconic acid

yellow red

Methodology

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

Preparation of Saponin Extracts

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 dichloro-methane

Concentra-ted saponins are extracted twice with an equal volume of n-butanol

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

Methodology

DNS Method

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 0.050 0.100 0.150 0.200

D-glucose calibration curve for determination of saponins

Glucose (0.4%)

and 500 μL of

DNS solution.

Measure at 540

nm.

Methodology

Sugar content in Quillaja saponin solutions

[Quillaja saponin] (mg/mL)

[Glu

cose

] (m

g/m

L)

Calibration curve of hydrolyzed sugars of Quillaja saponin

Hydrolysis of saponins (3-7

mg/mL) 5 mL of 6M HCl

+ 85°C pH 6.5-7.2

Sugar content is determined from

difference between hydrolyzed and non-hydrolyzed

saponins

Results

y = 7.08x + 0.024 R² = 1.00

0.0000

0.1000

0.2000

0.3000

0.4000

0.5000

0.6000

0.7000

0.0000 0.0200 0.0400 0.0600 0.0800 0.1000

Ab

sorb

an

ce

D-glucose concentration (mg/mL)

D-glucose calibration curve

Results

y = 0.156x - 0.001 R² = 0.718

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.000 0.050 0.100 0.150 0.200 0.250

Su

ga

r co

nce

ntr

ati

on

(m

g D

-g

luco

se/m

L)

Quillaja saponin concentration (mg/mL)

Sugar content in Quillaja saponin solutions

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

Plant Saponin concentration

(mg Quillaja

saponin/mL)

C. speciosus 0.0517

S. jambos 0.0655

T. spathacea -

T. anannassae 0.1714

Saponin concentrations in plant extracts

0.0000 0.0500 0.1000 0.1500 0.2000

C. speciosus

T.anannassae

S. jambos

T. spathacea

Saponin concentration (mg Quillaja saponin/mL)

Saponin concentrations in antidiabetic plant extracts

Results

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. Trease and Evans. Pharmacognosy. 1996. WB Saunders Company Limited, London, U.K.

2. 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).

3. Güçlü-Ünstündağ, Ö.; 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).

4. Rodríguez-Tirado, K.; Gavillán-Suárez J. University of Puerto Rico at Cayey, Cayey, P.R. Phytochemical studies of medicinal plants from several genera with antidiabetic properties. Unpublished work, 2011.

Thanks for your attention!

Spectrophotometric determination

of saponins in antidiabetic plant

extracts Natalie Rivera Ortiz

Mentor: Jannette Gavillán Suárez, Ph.D.

Department of Chemistry