Phytochemical screeningof medicinal plants fromseveral genera withantidiabetic activitiesKarla M. Rodríguez TiradoMentor: Dr.Jannette Gavillán- SuárezNovember 29th, 2011
Acknowledgements•Chemistry Department Technicians• Juan Carlos Rodríguez•Jannette Gavillán - Suárez, PhD
Phytochemicals and Diabetes Recent research have demonstrated the ability somephytochemicals have in protecting humans and of being usefulfor the treatment of diseases. Rupasinghe et al. (2003) have reported saponins antidiabeticproperties ( Kumar et al. 2009) Studies have also shown that glycosides, flavonoids, tannins andalkaloids have hypoglycemic activities ( Kumar et al. 2009) Terpenoids have also been shown to decrease blood sugar level inanimal studies (Kumar et al. 2009) Several plant derived flavonoids have been reported to inhibitaldose reductase activity and impart beneficial action in diabeticcomplications ( Tiwari and Rao 2002)
Our plants S.jambos- tannins, saponins,flavonoids and phenolic compoundshave been reported.(Djadjo et al. 2000,Reynertson et al,2008) Costus sp - previous studies revealthe occurrence of sterols,glycosides and saponins (da Silvaand Parente 2004) T. spathacea- has been reported tobe rich in flavonoids, triterpenesand 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 understudy Use thin layer chromatography (TLC) to identify thefollowing phytochemicals: alkaloids, flavonoids,terpenoids, tannins, saponins, phenolics, cardiacglycosides and sterols To compare the qualitative and TLC resultsSpecific Aims
RelevanceAt the moment, there is no phytochemical profile for theseplants to which people attribute antidiabetic properties. The chemical profile will allow the characterization of theherbal formulations (tea) use as complementary medicines. The chemical profile will allow to select specificphytochemicals and study their antidiabetic properties.
Methodology- QualitativeExperimentSterolsPrepare plantextractsTo the filtrate, add 0.5 ml ofacetic acid anhydride, 0.5 ml ofdichloromethane and 1 ml ofconcentrated sulphuric acid.A reddish brown ring willreveal a positive result
Methodology – QualitativeExperimentTerpenoidsDried methanolplant extractAdd 1 mL ofdichloromethaneand 2 ml ofconcentratedsulphuric acid.A reddish-brown color atthe interphaseindicates apositive result.
Methodology- TLCS. jambosT. anassaeT. spathaceaC. spiralisExtraction proceduresaccording to phytochemicalsSolvents:•CH2Cl2 and Methanol•Acetic Acid, Methanol andWater•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’sReagent•Iodine Vapors•Anasaldehyde-sulphuricacid• Vanillin- HCl Reagent
Results – FlavonoidsStandard: QuercetineExtraction: Methanol Liquid- liquid Extractionwith a mixture of water and ethyl acetateMobile Phase: CH2Cl2 and methanolVisualization: UV254nm and visible light
The TLC confirms thequalitative test result forTradescantia spathacea only .Except C. speciosus , all of theplants contains flavonoids.PlantT. spathacea +C. speciosus -S. jambos -T. anassae -
Results- AlkaloidsStandard: NicotineExtraction: NH4OH, lixiviate with EtOAc,add NH4OH to organic phase, extractwith CH2Cl2Mobile phase: CH2Cl2 and methanolVisualization: Spray withDragendorff’s reagent, UV254nm andvisible light
S. jambos, T. anassae and C.speciosus contains alkaloids.This results compare tothose obtain in thequalitative test.The TLC for T.spathaceaereveals the presence ofalkaloids, differing from thequalitative test result.Plant AlkaloidsT. spathacea -C. speciosus +S. jambos +T. anassae +
Results- PhenolicsStandard: HydroquinoneExtraction: Lixiviate with methanol and condense filtrateMobile phase: CH2Cl2 and methanolVisualization: Spray with Folin-Ciocalteu’s reagent and heat plates
The presence phenoliccompounds is observed in allplant extract.Tannins are phenolic compounds,therefore, this results confirm thoseobtained for the qualitative test inwhich every plant had a positiveresult. Literature confirms thepositive results for S. jambos andT.spathacea.Plant TanninsT. spathacea +C. speciosus +S. jambos +T .anassae +
Results: SterolsStandard: StigmasterolExtraction: Methanol and condense filtrateMobil phase: CH2Cl2, glacial acetic acid, methanol andwaterVisualization: Spray with a solution of FeCl3, acetic acidand sulfuric acid and heat the plate
Sterols are present in everyplant extract.The results confirms the qualitativetest results for T. spathacea, C.speciosus and T. anassae but not for S.jambos. Previous research haveconfirm the presence of sterols in C.speciosus.Plant SterolsT. spathacea +C. speciosus +S. jambos -T. anassae +
Results- Cardiac GlycosidesStandard: DigitoxinExtraction: 70%EtOH on rotary shaker, centrifuged 2times adding 70% lead acetate and 6.3% Na2CO3,respectively, redesolve with CH2Cl2Mobile phase: EtOAc-MeOH-H2OVisualization: Sulphuric Acid Reagent and UV366nm
The phytochemical is observedto be present in all of the plantextracts.Confirm the qualitative results for T.spathaceae, C. speciosus andT.anassae. This is not so for S. jambos,which had a negative for thequalitative. The presence of thisphytochemical in C. speciosus isrecorded by previous research.Plant CardiacGlycosidesT. spathacea +C. speciosus +S. jambos -T. anassae +
Results: SaponinsStandard: Commercial SaponinExtraction: Reflux leaves with 70% Ethanol , condense thefiltrate and add tert- butane.Mobile Phase: CH2Cl2, Glacial acetic acid, methanol and H2OVisualization: Anisaldehyde-sulphuric acid reagent , visiblelight and UV365nm
Saponin is present in all plantextracts.Plant SaponinsT. spathacea -C. speciosus -S. jambos +T. anassae -The results differ from the qualitativeresults in which only S. jambos gave apositive result. Literature confirmsthe presence of saponins in S. jambosand C. speciosus.
Results: TanninsStandard: Tannic AcidExtraction: Extract leaves with ethanol over nightMobile phase: Ethyl acetate and acetic acidVisualization: Vanillin-HCl Reagent
Plant TanninsT. spathacea +C. speciosus +S. jambos +T .anassae +Tannins are present inevery plant extract.This results confirm those obtainedfor the qualitative test in whichevery plant had a positive result.Tannins have been reported withhypoglycemic activities and to bepresent in S. jambos.
Results: TerpenoidsStandards: Ursolic Acid and StigmasterolExtractions: Powdered leaves extracted with methanol and water. Filtratewas acidified with sulphuric acid and extracted with dichloromethane.Mobile phase: Hexane: AcetoneVisualization: Anisaldehyde-sulphuric acid reagent , visible light and UV365nm
Plant TerpenoidsT. spathacea -C. speciosus -S. jambos +T. anassae -Terpenoids are found in everyplant extract.The results differ from the qualitative resultsin which only S. jambos gave a positive result.Terpenoids have been shown to decreaseblood 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 thanT.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 eachplant.
Conclusions The qualitative analysis did not demonstrate much exactitudewhen compared with the TLC analysis. Qualitative analysis gave false negatives to some plants thereforebeing less accurate than TLC and what reported in literature. TLC analysis showed the presence of the 8 phytochemicalsstudied in all the plants. Except C. speciosus Based on the results obtanied, specific TLC analysis could beused 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 spotsfor all the plants studied.
Future Work Isolate alkaloids, flavonoids and cardiac glycosides bycolumn cromatography. Perform HPLC analysis of fractions obtained by columnchromatography Complete the quantitave analysis of alkaloids in plantextracts.
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 futureprospects. Curr Sci, 2002, 83, 30–38. Mallikharjuna PB, Rajanna LN, Seetharam YN, Sharanabasappa GK. Phytochemical Studies of Strychnos potatoruna L.f.- AMedicinal 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 phenolicconstituents 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 ofliver 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. PhytochemicalsInvestigation on a Tropical Plant, Syzygium cumini from Kattuppalayam, Erode District, Tamil Nadu, South India. PakistanJournal of Nutrition 8 (1): 83-85, 2009.
Methodology – QualitativeExperimentFlavonoids – Shinoda TestDried methanolextracts of plants Add 95% methanol, drops ofconcentrated HCl and 0.5 g ofmagnessiumA pink color willreveal a positiveresult
Methodology – QualitativeExperimentAlkaloidsPrepare plantextractsTo the filtrate, adddrops of Wagner’sReagentA redddish-brownprecipitatesindicates a positiveresult