Phytochemical screening of medicinal plants from several genera with antidiabetic activities

Karla M. Rodrguez Tirado Mentor: Dr.Jannette Gavilln- Surez

November 29th, 2011

Acknowledgements

Chemistry Department Technicians Juan Carlos Rodrguez Jannette Gavilln - Surez, 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 (RosalesReyes et al., 2006 )

Goal
To assess the phytochemical profile of C. speciosus, T. anassae, S. jambos and T. spathacea

Specific Aims
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

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

Prepare plant extracts

Sterols
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 reddishbrown color at the interphase indicates a positive result.

Methodology- TLC
Extraction procedures according to phytochemicals

S. jambos T. anassae T. spathacea C. spiralis

UV264 and 366 Visible Light Dragendorffs Reagent Folin-Ciocalteus Reagent Iodine Vapors Anasaldehyde-sulphuric acid Vanillin- HCl Reagent

Solvents: CH2Cl2 and Methanol Acetic Acid, Methanol and Water CH2Cl2, glacial acetic acid, Water and Methanol Hexane and acetone Ethyl acetate and acetic acid

Qualitative Results
Plant Tan Alk Sap CG Ter Ste Fla

T. spathacea

+ +

+ +

+ +

+ +

+ -

C. speciosus

S. jambos
T. anassae

+
+
Tan Tannins Alk- Alkaloids Sap- Saponins Fla- Flavonoids

+
+

+
-

CG- Cardiac Glycosides Ter- Terpenoids Ste- Sterols

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

Plant T. spathacea C. speciosus

S. jambos T. anassae

+ -

The TLC confirms the qualitative test result for Tradescantia spathacea only .

Except C. speciosus , all of the plants contains flavonoids.

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 Dragendorffs reagent, UV254nm and visible light

Plant T. spathacea C. speciosus S. jambos T. anassae

Alkaloids + + +

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.

Results- Phenolics

Standard: Hydroquinone Extraction: Lixiviate with methanol and condense filtrate Mobile phase: CH2Cl2 and methanol Visualization: Spray with Folin-Ciocalteus reagent and heat plates

The presence phenolic compounds is observed in all plant extract. Plant T. spathacea Tannins +

C. speciosus S. jambos T .anassae

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.

+ + +

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.

Plant T. spathacea C. speciosus S. jambos T. anassae 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.

Sterols + + +

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

Plant T. spathacea C. speciosus S. jambos T. anassae

Cardiac Glycosides + + +

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.

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

Plant T. spathacea C. speciosus S. jambos T. anassae

Saponins + -

Saponin is present in all plant extracts.

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 T. spathacea C. speciosus S. jambos T .anassae

Tannins + + + + 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 T. spathacea C. speciosus S. jambos T. anassae

Terpenoids + -

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, 3038. 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, Delme 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., Rodrguez-Mendiola, M., Fattel-Fazenda, S., Arce-Popoca, E., HernndezGarca, S., Villa-Trevio, 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 Fr 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

Alkaloids

Tannins
Cardiac

Glycosides

Sterols Phenols

Terpenoids

Flavonoids

Saponins

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 Wagners Reagent A redddish-brown precipitates indicates a positive result