Reynaldo J. Morales RodriguezYadira D. Cora AmaroElsa M. Luciano NunezClaudia A. Ospina, Ph.D.Mayra Pagán Ortiz, Ph.D.
 Family: Zygophyllaceae Genus: Guaiacum Species: Officinale Common name:Guayacán, Palo Santo,Lignum Vitae Distributio...
 Family:Simaroubaceae Commonly Knownas “aceitillo falso” Trees of 2-8 meters Distribution: Plantendemic of PuertoRico ...
Simaroubacea family Traditional Uses: Anti-malaria Anti-feedant Anti-inflammatory Anti-leukemic Anti-viralSimarouba ...
Metabolites isolated from other species ofSimaroubacea family• Quassinoids– Group of highly oxygenated terpenes– Responsib...
 To expand to the phytomedicinal knowledge of nativeand endemic plants of Puerto Rico and to discovertheir chemotaxonomy....
Selection of theorganismCollection of theorganismPreparation ofthe crude extractBiological testPurification ofchemicalcons...
Plant CollectionJayuyaGuaiacum Officinale•1.12kg de hojas•98.5 g extractocrudo
Extraction
1H NMR Spectrum (400MHz) of crude extract in CDCL3Alyphatic
1H NMR Spectrum (400MHz) of CHCl3 in CDCL3
* All extracts were evaluated at a single dose of 100 μg/mL.** Results performed by Dr. Marianela Pérez Torres in the UPR-...
 First collection Place: Patillas P.R. 2008 Extractions
Table 2. Dry weight for crude extracts and solvent usedfor each extraction in the first collectionPlant Extract Extract dr...
Table 3. Cytotoxicity results for Simarouba tulae leaves extractsagainst Artemia Salina testSimarouba tulaeExtractArtemia ...
GuayacanAqueous LayerExtraction:•ChloroformAqueouslayerOrganiclayerRotoevaporationTLCDrying atRoomTemp. hexane/ethylacetat...
• Chloroform /Methanol(9.8:0.2)• Hexane/Ethyl Acetate (9:1)TLC
 Second collection Place: Maricao P.R. 2009 Preparation of the extractsTable 6. Dry weight for crude extracts in the se...
Table 7. Cytotoxicity results for Simarouba tulae leaves extractsSimaroubatulaeExtractArtemiaSalina TestLC50 value inµg/mL...
Table 7. Cytotoxicity results for Simarouba tulae leaves extractsSimaroubatulaeExtractArtemiaSalina TestLC50 value inµg/mL...
Table 7. Cytotoxicity results for Simarouba tulae leaves extractsSimaroubatulaeExtractArtemiaSalina TestLC50 value inµg/mL...
Chloroform extract (40g)
Chloroform extract (40g)CC Si gel (95:5 CHCl3/MeOH)
Chloroform extract (40g)CC Si gel (95:5 CHCl3/MeOH)28 fractionsNMR AnalysisTLC(9.8:0.2 CHCl3/MeOH)
Chloroform extract (40g)CC Si gel (95:5 CHCl3/MeOH)28 fractionsSH2C3CC Lipophilic Sephadex(95:5 CHCl3/MeOH)
Chloroform extract (40g)CC Si gel (95:5 CHCl3/MeOH)28 fractionsSH2C3CC Lipophilic Sephadex(95:5 CHCl3/MeOH)SH2C3CTLC(97:3)...
Chloroform extract (40g)CC Si gel (95:5 CHCl3/MeOH)28 fractionsSH2C3CC Lipophilic Sephadex(95:5 CHCl3/MeOH)SH2C3CSH2C3C-C
AliphaticAllylicAnalysis using NMR spectroscopyFigure 4. 1H NMR Spectrum (400 MHz) of SH2C3C-C in CDCl3α-HeteroatomsVinylic
Analysis using NMR spectroscopyFigure 4. 13C NMR Spectrum (100 MHz) of SH2C3C-C in CDCl3AliphaticOxygenatedVinylicCarbonyls
Hexane extract (31g)
Hexane extract (31g)CC Si gel (CHCl3/Acetona)
Hexane extract (31g)CC Si gel (CHCl3/Acetona)≈ 23 fractions
1H NMR Spectrum (400 MHz) for crude extract of the leavesof S. tulaeAlkenesOxygenated C’sC-C σ bonds
• Every extract of Guaiacum officinale presentedactivity in the Artemia Salina bioassay.• From spectroscopic chloroform ex...
 The chloroform extract of Simarouba tulae leaves showedhigh cytotoxic activity against Artemia Salina and twobreast canc...
• Identification of the main compound of theChloroform extract using NMR spectroscopy.• Perform a Chromatographic analysis...
 Batista, J.; Braz, R.; Curcino, I.; Da Silva, M.; Rodrigues E.; Vireira, P. “20(R)- and 20(S)- SimarolideEpimers Isolate...
 Advising Claudia Ospina, PhD Mayra Pagan, PhD Financial Support Dean of Academic Affairs Undergraduate students Os...
Reynaldo J. Morales RodriguezYadira D. Cora AmaroElsa M. Luciano NunezClaudia A. Ospina, Ph.D.Mayra Pagán Ortiz, Ph.D.
Data CollectionCount the death shrimps Add MeOH Count the total shrimpsBioassayPrepare the concentrationassigned to each l...
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Ciano yadira cora-reynaldo-morales_28nov2011

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Ciano yadira cora-reynaldo-morales_28nov2011

  1. 1. Reynaldo J. Morales RodriguezYadira D. Cora AmaroElsa M. Luciano NunezClaudia A. Ospina, Ph.D.Mayra Pagán Ortiz, Ph.D.
  2. 2.  Family: Zygophyllaceae Genus: Guaiacum Species: Officinale Common name:Guayacán, Palo Santo,Lignum Vitae Distribution: Native toWest Indies and SouthAmerica Historical uses: Gout,syphilis, arthritis, resinused to detect blood inhuman stool.
  3. 3.  Family:Simaroubaceae Commonly Knownas “aceitillo falso” Trees of 2-8 meters Distribution: Plantendemic of PuertoRico (Maricao andPatillas)Simarouba tulae
  4. 4. Simaroubacea family Traditional Uses: Anti-malaria Anti-feedant Anti-inflammatory Anti-leukemic Anti-viralSimarouba tulae
  5. 5. Metabolites isolated from other species ofSimaroubacea family• Quassinoids– Group of highly oxygenated terpenes– Responsible for its therapeutic properties– Taxonomic markerBasic skeleton of a quassinoid C-20
  6. 6.  To expand to the phytomedicinal knowledge of nativeand endemic plants of Puerto Rico and to discovertheir chemotaxonomy. Isolate, purify and identify chemical compounds ofGuaiacum officinale and Simarouba tulae leaves. Evaluate the cytotoxic activity of pure compounds ofGuaiacum officinale and Simarouba tulae leaves.
  7. 7. Selection of theorganismCollection of theorganismPreparation ofthe crude extractBiological testPurification ofchemicalconstituents
  8. 8. Plant CollectionJayuyaGuaiacum Officinale•1.12kg de hojas•98.5 g extractocrudo
  9. 9. Extraction
  10. 10. 1H NMR Spectrum (400MHz) of crude extract in CDCL3Alyphatic
  11. 11. 1H NMR Spectrum (400MHz) of CHCl3 in CDCL3
  12. 12. * All extracts were evaluated at a single dose of 100 μg/mL.** Results performed by Dr. Marianela Pérez Torres in the UPR-MSC.*** LC50 values greater than 200 µg/ml are not considered cytotoxicPlant species Extract LC50 inµg/mL fromthe BrineShrimpLethalityTest% of growth inhibition‡ onvarious breast cancer celllinesMCF-7 ZR-75-1GuaiacumoficinaleCrude 26.125 81 80Hexane 30.765 - -Chloroform 0.692 91 76Ethyl Acetate 4.479 - -Table 1. Cytotoxicity results for Simarouba tulae leaves extracts
  13. 13.  First collection Place: Patillas P.R. 2008 Extractions
  14. 14. Table 2. Dry weight for crude extracts and solvent usedfor each extraction in the first collectionPlant Extract Extract dry weight(g) ±0.02Simarouba tulae Crude 15.0Hexane 2.33Chloroform 9.21Ethyl acetate 0.90
  15. 15. Table 3. Cytotoxicity results for Simarouba tulae leaves extractsagainst Artemia Salina testSimarouba tulaeExtractArtemia Salina TestLC50 value in µg/mLᶱCrude 2Hexane > 200Chloroform 161Ethyl acetate 35
  16. 16. GuayacanAqueous LayerExtraction:•ChloroformAqueouslayerOrganiclayerRotoevaporationTLCDrying atRoomTemp. hexane/ethylacetate (9:1)chloroform/methanol(9.8:0.2)ExtractGuayacan aqueous layer
  17. 17. • Chloroform /Methanol(9.8:0.2)• Hexane/Ethyl Acetate (9:1)TLC
  18. 18.  Second collection Place: Maricao P.R. 2009 Preparation of the extractsTable 6. Dry weight for crude extracts in the second collectionof Simarouba tulaeExtractSimarouba tulaeExtract dry weight (g)±0.02Crude 113.00Hexane 30.88Chloroform 39.64Ethyl Acetate 6.08
  19. 19. Table 7. Cytotoxicity results for Simarouba tulae leaves extractsSimaroubatulaeExtractArtemiaSalina TestLC50 value inµg/mLᶱBreast Cancer CellGrowth inhibition % *ᵜMCF-7 ZR-75-1 T47DCrude 23.703 82 < 80 94Hexane > 200 87 < 80 92Chloroform 157.141 95 < 80 97Ethyl acetate 26.243 < 80 < 80 92
  20. 20. Table 7. Cytotoxicity results for Simarouba tulae leaves extractsSimaroubatulaeExtractArtemiaSalina TestLC50 value inµg/mLᶱBreast Cancer CellGrowth inhibition % *ᵜMCF-7 ZR-75-1 T47DCrude 23.703 82 < 80 94Hexane > 200 87 < 80 92Chloroform 157.141 95 < 80 97Ethyl acetate 26.243 < 80 < 80 92
  21. 21. Table 7. Cytotoxicity results for Simarouba tulae leaves extractsSimaroubatulaeExtractArtemiaSalina TestLC50 value inµg/mLᶱBreast Cancer CellGrowth inhibition % *ᵜMCF-7 ZR-75-1 T47DCrude 23.703 82 < 80 94Hexane > 200 87 < 80 92Chloroform 157.141 95 < 80 97Ethyl acetate 26.243 < 80 < 80 92
  22. 22. Chloroform extract (40g)
  23. 23. Chloroform extract (40g)CC Si gel (95:5 CHCl3/MeOH)
  24. 24. Chloroform extract (40g)CC Si gel (95:5 CHCl3/MeOH)28 fractionsNMR AnalysisTLC(9.8:0.2 CHCl3/MeOH)
  25. 25. Chloroform extract (40g)CC Si gel (95:5 CHCl3/MeOH)28 fractionsSH2C3CC Lipophilic Sephadex(95:5 CHCl3/MeOH)
  26. 26. Chloroform extract (40g)CC Si gel (95:5 CHCl3/MeOH)28 fractionsSH2C3CC Lipophilic Sephadex(95:5 CHCl3/MeOH)SH2C3CTLC(97:3) CHCl3/MeOH)CC Si gel(95:5 CHCl3/MeOH)
  27. 27. Chloroform extract (40g)CC Si gel (95:5 CHCl3/MeOH)28 fractionsSH2C3CC Lipophilic Sephadex(95:5 CHCl3/MeOH)SH2C3CSH2C3C-C
  28. 28. AliphaticAllylicAnalysis using NMR spectroscopyFigure 4. 1H NMR Spectrum (400 MHz) of SH2C3C-C in CDCl3α-HeteroatomsVinylic
  29. 29. Analysis using NMR spectroscopyFigure 4. 13C NMR Spectrum (100 MHz) of SH2C3C-C in CDCl3AliphaticOxygenatedVinylicCarbonyls
  30. 30. Hexane extract (31g)
  31. 31. Hexane extract (31g)CC Si gel (CHCl3/Acetona)
  32. 32. Hexane extract (31g)CC Si gel (CHCl3/Acetona)≈ 23 fractions
  33. 33. 1H NMR Spectrum (400 MHz) for crude extract of the leavesof S. tulaeAlkenesOxygenated C’sC-C σ bonds
  34. 34. • Every extract of Guaiacum officinale presentedactivity in the Artemia Salina bioassay.• From spectroscopic chloroform extracts of Guaiacumofficinale are rich in metabolites.
  35. 35.  The chloroform extract of Simarouba tulae leaves showedhigh cytotoxic activity against Artemia Salina and twobreast cancer cell lines. From spectroscopic data SH2C3C-C is rich in metabolites The hexane extract of Simarouba tulae leaves showed highcytotoxic activity against two breast cancer cell lines.
  36. 36. • Identification of the main compound of theChloroform extract using NMR spectroscopy.• Perform a Chromatographic analysis ofChloroform• To evaluate the cytotoxicity of purecompounds against cancer cell lines.
  37. 37.  Batista, J.; Braz, R.; Curcino, I.; Da Silva, M.; Rodrigues E.; Vireira, P. “20(R)- and 20(S)- SimarolideEpimers Isolated from Simaba cuneata Chemical Shifts Assignment of Carbon and Hydrogen Atoms”. J.Braz. Chem. Soc., 1999, 10, 76-84. Beutler, J.; Clement, J.; Goncharova, E.; et al. “ Quassinoid Inhibition of AP-1 Function does notCorrelate with Cytotoxicity or Protein Synthesis Inhibition”. Journal of Natural Products, 2009 Anderson, M.; Gupta, M.; Phillipson, D.; Solis, P.; Colin, W. “A Microwell Cytotoxicity Assay usingArtemia Salina (Brine Shrimp)”. Planta Med, 1993, 59, 250-252. Guo, Z.; Sindelar, R.D.; Sindelar, R.W.; Vangapandu, S.; Walker, L.A. Biological Actives Quassinoids andTheir Chemistry: Potential Leads for Drug Design. Curr. Med. Chem. 2005, 12, 173-190. Rhodes, M.; Robins, R. High-performance liquid chromatographic methods for the analysis andpurification of quassinoids from Quassia amara L. J. Chromato. 1984, 283, 436-440. Ospina, C. A.; Pagán, M.; Carvajal, A.; Claudio, K; Rivera, J.; Ortiz, I.; Hernández, J. In “CytotoxicScreening of Tropical Plants Using Brine Shrimp Lethality Test”.; Montes, E. L.; Eds.; Cuadernos deInvestigación Number 7; Instituto de Investigaciones Interdisciplinarias: Cayey, 2009; 1-20.
  38. 38.  Advising Claudia Ospina, PhD Mayra Pagan, PhD Financial Support Dean of Academic Affairs Undergraduate students Ospina and Pagan’s research group Technical Support Chemistry Department Melvin De Jesus, UPR Humacao Collaborators Augusto Carvajal, M.S Marianela Pérez – School of PharmacyUPR Karla Claudio- Graduate student Janibeth Hernández-Graduate student
  39. 39. Reynaldo J. Morales RodriguezYadira D. Cora AmaroElsa M. Luciano NunezClaudia A. Ospina, Ph.D.Mayra Pagán Ortiz, Ph.D.
  40. 40. Data CollectionCount the death shrimps Add MeOH Count the total shrimpsBioassayPrepare the concentrationassigned to each lineAdd 10-15 brine shrimps bypippetingIncubate for 24 hoursSamples PreparationPositive Control: BerberineChlorideNegative Control: SalineSolutionPlants extractions dissolved inDMSOBrine shrimp incubationSpecialized recipientIncubate @ 22-29 ◦C for 48hoursLarvae emerges byphototropic effectSaline SolutionYeast Marine salt Distilled water

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