spectrophotometric determination of saponins in antidiabetic plant extracts. natalie rivera....
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Natalie Rivera. Mentor(a): Jannette GavillánTRANSCRIPT
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Spectrophotometric determination
of saponins in antidiabetic plant
extracts Natalie Rivera Ortiz
Mentor: Jannette Gavillán Suárez, Ph.D.
Department of Chemistry
May 2012
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Acknowledgements
• Dr. Jannette Gavillán, Ph.D.
• Chemistry Department Laboratory Technicians
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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).
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Saponins
Two kinds of saponins:
Quillaja saponin
Pentacyclic triterpenoid saponins Steroidal saponins
Digitonin
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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
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• 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
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Our plants
Costus speciosus Tradescantia
spathacea
Syzygium jambos Tapeinochilus anannassae
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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.
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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//
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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
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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
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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
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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.
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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
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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
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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
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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
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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
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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
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Current work
• Optimization of hydrolysis conditions and
Quillaja saponin calibration curve .
• Determination of saponin content in T.
spathacea extracts.
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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.
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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.
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Thanks for your attention!
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Spectrophotometric determination
of saponins in antidiabetic plant
extracts Natalie Rivera Ortiz
Mentor: Jannette Gavillán Suárez, Ph.D.
Department of Chemistry