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Año 2011 - 2012Recursos:Natalie Rivera OrtizJannette Gavillán Ph.D.
Citation preview
Quantitative analysis of
phytochemicals in antidiabetic
plant extracts
Natalie Rivera Ortiz
Mentor: Dr. Jannette Gavillán Suárez, Ph.D
Department of Chemistry
CHEM -4999
Acknowledgements
Thanks to:
Dr. Jannette Gavillán Suárez, 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
Ursolic Acid Isoprene
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 μg/mL)
stimulated an increase of 228% in insulin release from
rat pancreatic β-cells compared to the control (5.6 mM
glucose).7
Goal and Objectives
Objectives:
Quantify flavonoids, total phenolic compounds
and saponins in plant extracts from Tapeinochuilus
annanassae, Syzygium jambos, Costus speciosus, and
Tradescantia spathacea.
Goal:
Quantify possible biomarkers of antidiabetic
activity of plant extracts from several genera.
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
μL of AlCl3
10%and 200 μL
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 μL
of Lowry
reactive C and
200 μL of Lowry
reactive E
Quercetin
Calibration
Curve
Measure
absorbance at
760 nm and
determine
concentration.
Results - Flavonoids
y = 34.8x - 0.055 R² = 0.994
0.0000
0.1000
0.2000
0.3000
0.4000
0.5000
0.6000
0.7000
0.8000
0.00E+00 5.00E-03 1.00E-02 1.50E-02 2.00E-02 2.50E-02
Ab
sorb
an
ce (
415
nm
)
[Quercetin] (mg/mL)
Calibration curve for determination of flavonoids in antidiabetic plant extracts
Results - Flavonoids
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
Calculations
Calculations
Results - Flavonoids
Flavonoid content by dry weight:
(0.0224 mg/mL QE)(1mL) = 29.8 mg QE/g DW
Flavonoid content by dry weight:
(0.0224 mg/mL QE)(1mL) = 29.8 mg QE/g DW
0.000819 g DW
Results - Flavonoids
Plant Concentration
of flavonoids
(± SD mg
QE/ g DW)
T. spathacea 13.7 ± 0.7
C. speciosus 15.8 ± 0.9
S. jambos 24.3 ± 1.3
T. anannassae 29.8 ± 0.0 0 5 10 15 20 25 30 35
S. jambos
T. anannassae
C. speciosus
T. spathacea
[Flavonoid] (mgQE/g DW)
Pla
nt
Flavonoid content in antidiabetic plants
Results – Total Phenols
y = 4.63x + 0.16 R² = 0.990
0.0000
0.1000
0.2000
0.3000
0.4000
0.5000
0.6000
0.7000
0.8000
0.9000
1.0000
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18
Ab
sorb
an
ce (
760 n
m)
Quercetin concentration (mg/mL)
Calibration curve for determination of total phenolic content
Results – Total Phenols
Concentration of
methanolic extract in
assay:
C1V1=C2V2
C2 = C1V1/V2
C2 = (0.150μL)(3.00 mg/mL)
(1500 μL)
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
Calculations
Results – Total Phenols
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)
Calculations
Results – Total Phenols
Aqueous
extract
Total
Phenolic
Content
(± SD mg
QE)
T. spathacea 0.26 ± 0.01
C. speciosus 0.35 ± 0.01
S. jambos 0.45 ± 0.09
T. anannassae 0.14 ± 0.02
Methanolic
extract
Total
Phenolic
Content
(± SD mg
QE)
T. spathacea 0.052 ± 0.002
C. speciosus 0.060 ± 0.002
S. jambos 0.18 ± 0.04
T. anannassae 0.034 ± 0.005
Results – Total Phenols
0 5 10 15
S. jambos
T.anannassae
C. speciosus
T. spathacea
Total Phenolic Content (mg QE/mg extract)
Pla
nt
Total Phenolic Content in Aqueous and Methanolic Extracts
aqueous
methanolic
Current Work
Concentrated
saponins are
extracted twice
with equal
volumes of
n-butanol
DNS Method Quantitative
determination
of glucose.
Glucose (0.4%)
and 500 μL of
DNS solution.
Calibration
curve of D-
glucose
(540 nm)
3,5-dinitrosalicylic
acid D-glucose 3-amino-5-
nitrosalicylic acid
D-gluconic
acid
Reaction:
Current Work
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 0.020 0.040 0.060 0.080 0.100 0.120 0.140 0.160 0.180
Ab
sorb
an
ce
Concentration (mg/mL)
D-glucose calibration curve for determination of saponins
Current Work
Sugar content in
Quijalla saponin
is determined
from difference
between
hydrolyzed and
non-hydrolyzed
saponins
Quillaja saponin
Sugar content in Quillaja
saponin solutions
[Quillaja saponin] (mg/mL)
[Glu
cose
] (m
g/m
L)
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. Rodríguez-Tirado, K.; Gavillán-Suárez. 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 Biolo-
gical Significance. Int.J.Mol.Sci. [Online] 2008, 8, 950-988.
http://www.mdpi.org// (accessed March 8, 2011).
3. Güçlü-Ünstündağ, Ö.; Mazza, G. Saponins: properties, applica-
tions 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.;
Araújo, 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-induced-
diabetic 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 Gavillán Suárez, Ph.D
Department of Chemistry
CHEM -4999