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Natalie Rivera. Mentor(a): Jannette Gavillán
Citation preview
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