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.

Leach MJ. Gymnema sylvestre for Diabetes Mellitus: A Systematic Review. J Altern Complement Med. 2007 Nov; 13(9):977-83

Tiwari AK, Rao JM: Diabetes mellitus and multiple therapeutic approaches of phytochemicals: present status and future

prospects. Curr Sci, 2002, 83, 30–38.

Mallikharjuna PB, Rajanna LN, Seetharam YN, Sharanabasappa GK. Phytochemical Studies of Strychnos potatoruna L.f.- A

Medicinal Plant. Journal of Chemistry. 4 (4): 510-518, 2007.

Djadjo C, Delmée M, Quentin-Leclercq J, (2000). Antimicrobial activity of bark extracts of Syzygium jambos(L.)

Alston(Myrtacea). Journal of Ethnopharmacology, Volume 71, Issues 1-2, July 2000, Pages 307-313

Reynertson, K. A., Yang, H., Jiang, B., Basile, M. J., & Kennelly, E. J. (2008). Quantitative analysis of antiradical phenolic

constituents from fourteen edible Myrtaceae fruits. Food Chemistry, 109(4), 883-890. doi:10.1016/j.foodchem.2008.01.021

Rosales-Reyes, T., de la Garza, M., Arias-Castro, C., Rodríguez-Mendiola, M., Fattel-Fazenda, S., Arce-Popoca, E., Hernández-

García, S., Villa-Treviño, S.Aqueous crude extract of Rhoeo discolor, a Mexican medicinal plant, decreases the formation of

liver preneoplastic foci in rats. Journal of Ethnopharmacology, Volume 115, Issue 3, 12 February 2008, Pages 381-386

da Silva, B., & Parente, J. (2004). New steroidal saponins from rhizomes of Costus spiralis. Zeitschrift Für Naturforschung. C,

Journal Of Biosciences, 59(1-2), 81-85. Retrieved from EBSCOh

Kumar A, Ilavarasan R, Jayachandran T, Decaraman M, Aravindhan P , Padmanabhan N, Krishnan MRV. Phytochemicals

Investigation on a Tropical Plant, Syzygium cumini from Kattuppalayam, Erode District, Tamil Nadu, South India. Pakistan

Journal of Nutrition 8 (1): 83-85, 2009.

Thanks for the attention…

Cardiac

Glycosides

Terpenoids Flavonoids

Alkaloids

Phenols

Saponins

Sterols

Tannins

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

Methodology – Qualitative

Experiment

Alkaloids

Prepare plant

extracts

To the filtrate, add

drops of Wagner’s Reagent

A redddish-brown

precipitates

indicates a positive

result