Phytochemical screening of medicinal plants from several genera with antidiabetic activities Karla M. Rodríguez Tirado Mentor: Dr.Jannette Gavillán- Suárez November 29 th , 2011
Phytochemical screening
of medicinal plants from
several genera with
antidiabetic activities
Karla M. Rodríguez Tirado
Mentor: Dr.Jannette Gavillán- Suárez
November 29th, 2011
Acknowledgements
•Chemistry Department Technicians
• Juan Carlos Rodríguez
•Jannette Gavillán - Suárez, PhD
Phytochemicals and Diabetes
Recent research have demonstrated the ability some
phytochemicals have in protecting humans and of being useful
for the treatment of diseases.
Rupasinghe et al. (2003) have reported saponins antidiabetic
properties ( Kumar et al. 2009)
Studies have also shown that glycosides, flavonoids, tannins and
alkaloids have hypoglycemic activities ( Kumar et al. 2009)
Terpenoids have also been shown to decrease blood sugar level in
animal studies (Kumar et al. 2009)
Several plant derived flavonoids have been reported to inhibit
aldose reductase activity and impart beneficial action in diabetic
complications ( Tiwari and Rao 2002)
Our plants S.jambos- tannins, saponins,
flavonoids and phenolic compounds
have been reported.
(Djadjo et al. 2000,Reynertson et al,
2008)
Costus sp - previous studies reveal
the occurrence of sterols,
glycosides and saponins (da Silva
and Parente 2004)
T. spathacea- has been reported to
be rich in flavonoids, triterpenes
and phenolics compounds (Rosales-
Reyes et al., 2006 )
Goal To assess the phytochemical profile of C. speciosus, T.
anassae, S. jambos and T. spathacea
To complete the qualitative analysis of alkaloids,
flavonoids, sterols and terpenoids for the plants under
study
Use thin layer chromatography (TLC) to identify the
following phytochemicals: alkaloids, flavonoids,
terpenoids, tannins, saponins, phenolics, cardiac
glycosides and sterols
To compare the qualitative and TLC results
Specific Aims
Relevance
At the moment, there is no phytochemical profile for these
plants to which people attribute antidiabetic properties.
The chemical profile will allow the characterization of the
herbal formulations (tea) use as complementary medicines.
The chemical profile will allow to select specific
phytochemicals and study their antidiabetic properties.
Methodology- Qualitative
Experiment
Sterols
Prepare plant
extracts
To the filtrate, add 0.5 ml of
acetic acid anhydride, 0.5 ml of
dichloromethane and 1 ml of
concentrated sulphuric acid.
A reddish brown ring will
reveal a positive result
Methodology – Qualitative
Experiment
Terpenoids
Dried methanol
plant extract
Add 1 mL of
dichloromethane
and 2 ml of
concentrated
sulphuric acid. A reddish-
brown color at
the interphase
indicates a
positive result.
Methodology- TLC
S. jambos
T. anassae
T. spathacea
C. spiralis
Extraction procedures
according to phytochemicals
Solvents:
•CH2Cl2 and Methanol
•Acetic Acid, Methanol and
Water
•CH2Cl2, glacial acetic acid,
Water and Methanol
• Hexane and acetone
•Ethyl acetate and acetic acid
•UV264 and 366
•Visible Light
•Dragendorff’s Reagent
•Folin-Ciocalteu’s Reagent
•Iodine Vapors
•Anasaldehyde-sulphuric
acid
• Vanillin- HCl Reagent
Qualitative Results
Plant
Tan
Alk
Sap
CG
Ter
Ste
Fla
T. spathacea + - - + + + +
C. speciosus + + - + + + -
S. jambos + + + - - - -
T. anassae + + - + - + -
Tan –Tannins CG- Cardiac Glycosides
Alk- Alkaloids Ter- Terpenoids
Sap- Saponins Ste- Sterols
Fla- Flavonoids
Results – Flavonoids
Standard: Quercetine
Extraction: Methanol Liquid- liquid Extraction
with a mixture of water and ethyl acetate
Mobile Phase: CH2Cl2 and methanol
Visualization: UV254nm and visible light
The TLC confirms the
qualitative test result for
Tradescantia spathacea only .
Except C. speciosus , all of the
plants contains flavonoids.
Plant
T. spathacea +
C. speciosus -
S. jambos -
T. anassae -
Results- Alkaloids
Standard: Nicotine
Extraction: NH4OH, lixiviate with EtOAc,
add NH4OH to organic phase, extract
with CH2Cl2
Mobile phase: CH2Cl2 and methanol
Visualization: Spray with
Dragendorff’s reagent, UV254nm and visible light
S. jambos, T. anassae and C.
speciosus contains alkaloids.
This results compare to
those obtain in the
qualitative test.
The TLC for T.spathaceae
reveals the presence of
alkaloids, differing from the
qualitative test result.
Plant Alkaloids
T. spathacea -
C. speciosus +
S. jambos +
T. anassae +
Results- Phenolics
Standard: Hydroquinone
Extraction: Lixiviate with methanol and condense filtrate
Mobile phase: CH2Cl2 and methanol
Visualization: Spray with Folin-Ciocalteu’s reagent and heat plates
The presence phenolic
compounds is observed in all
plant extract.
Tannins are phenolic compounds,
therefore, this results confirm those
obtained for the qualitative test in
which every plant had a positive
result. Literature confirms the
positive results for S. jambos and
T.spathacea.
Plant Tannins
T. spathacea +
C. speciosus +
S. jambos +
T .anassae +
Results: Sterols
Standard: Stigmasterol
Extraction: Methanol and condense filtrate
Mobil phase: CH2Cl2, glacial acetic acid, methanol and
water
Visualization: Spray with a solution of FeCl3, acetic acid
and sulfuric acid and heat the plate
Sterols are present in every
plant extract.
The results confirms the qualitative
test results for T. spathacea, C.
speciosus and T. anassae but not for S.
jambos. Previous research have
confirm the presence of sterols in C.
speciosus.
Plant Sterols
T. spathacea +
C. speciosus +
S. jambos -
T. anassae +
Results- Cardiac Glycosides
Standard: Digitoxin
Extraction: 70%EtOH on rotary shaker, centrifuged 2
times adding 70% lead acetate and 6.3% Na2CO3,
respectively, redesolve with CH2Cl2
Mobile phase: EtOAc-MeOH-H2O
Visualization: Sulphuric Acid Reagent and UV366nm
The phytochemical is observed
to be present in all of the plant
extracts.
Confirm the qualitative results for T
.spathaceae, C. speciosus and
T.anassae. This is not so for S. jambos,
which had a negative for the
qualitative. The presence of this
phytochemical in C. speciosus is
recorded by previous research.
Plant Cardiac
Glycosides
T. spathacea +
C. speciosus +
S. jambos -
T. anassae +
Results: Saponins
Standard: Commercial Saponin
Extraction: Reflux leaves with 70% Ethanol , condense the
filtrate and add tert- butane.
Mobile Phase: CH2Cl2, Glacial acetic acid, methanol and H2O
Visualization: Anisaldehyde-sulphuric acid reagent , visible
light and UV365nm
Saponin is present in all plant
extracts.
Plant Saponins
T. spathacea -
C. speciosus -
S. jambos +
T. anassae -
The results differ from the qualitative
results in which only S. jambos gave a
positive result. Literature confirms
the presence of saponins in S. jambos
and C. speciosus.
Results: Tannins
Standard: Tannic Acid
Extraction: Extract leaves with ethanol over night
Mobile phase: Ethyl acetate and acetic acid
Visualization: Vanillin-HCl Reagent
Plant Tannins
T. spathacea +
C. speciosus +
S. jambos +
T .anassae +
Tannins are present in
every plant extract.
This results confirm those obtained
for the qualitative test in which
every plant had a positive result.
Tannins have been reported with
hypoglycemic activities and to be
present in S. jambos.
Results: Terpenoids
Standards: Ursolic Acid and Stigmasterol
Extractions: Powdered leaves extracted with methanol and water. Filtrate
was acidified with sulphuric acid and extracted with dichloromethane.
Mobile phase: Hexane: Acetone
Visualization: Anisaldehyde-sulphuric acid reagent , visible light and UV365nm
Plant Terpenoids
T. spathacea -
C. speciosus -
S. jambos +
T. anassae -
Terpenoids are found in every
plant extract.
The results differ from the qualitative results
in which only S. jambos gave a positive result.
Terpenoids have been shown to decrease
blood sugar level in animal studies.
Summary Flavonoids:
Costus speciosus was the only plant without the phytochemical.
Alkaloids:
The presence of alkaloids was observed for all plants studied
R. spathaceae and C.spiralis showed higher amount of spots than
T.ananassae and S.jambos.
Phenolics:
The presence of phenolics was observed for all plants studied
S.jambos presents the most amount of spots this phytochemical.
Sterols, Cardiac Glycosides, Saponins, Tannins and Terpenoids
Their presence was observed for all plants studied
The amount of spots for each phytochemical was similar for each
plant.
Conclusions
The qualitative analysis did not demonstrate much exactitude
when compared with the TLC analysis.
Qualitative analysis gave false negatives to some plants therefore
being less accurate than TLC and what reported in literature.
TLC analysis showed the presence of the 8 phytochemicals
studied in all the plants.
Except C. speciosus
Based on the results obtanied, specific TLC analysis could be
used to characterize plant extracts.
Conclusions
TLC analysis chosen for plant characterization:
S. jambos: Cardiac glycosides (orange spot)
C. speciosus: alkaloid (amount of spots)
T. spathacea: alkaloids (mint green colored spot), flavonoids
(amount of spots)
T. anassae: alkaloids (light blue spot)
TLC for phenolics and terpenoinds showed the most spots
for all the plants studied.
Future Work
Isolate alkaloids, flavonoids and cardiac glycosides by
column cromatography.
Perform HPLC analysis of fractions obtained by column
chromatography
Complete the quantitave analysis of alkaloids in plant
extracts.
References Wagner R and Bladt S, Plant Drug Analysis, A Thin Layer Chromatography Atlas, 2nd Ed: Springer;Berlin, 1996.
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Methodology – Qualitative
Experiment
Flavonoids – Shinoda Test
Dried methanol
extracts of plants Add 95% methanol, drops of
concentrated HCl and 0.5 g of
magnessium
A pink color will
reveal a positive
result