Reynaldo J. Morales Rodriguez Yadira D. Cora Amaro Elsa M. Luciano Nunez Claudia A. Ospina, Ph.D.

Mayra Pagn Ortiz, Ph.D.

Family: Zygophyllaceae Genus: Guaiacum Species: Officinale Common name: Guayacn, Palo Santo, Lignum Vitae Distribution: Native to West Indies and South America Historical uses: Gout, syphilis, arthritis, resin used to detect blood in human stool.

Simarouba tulae

Family:

Simaroubaceae
Commonly

Known as aceitillo falso of 2-8 meters

Trees

Distribution:

Plant endemic of Puerto Rico (Maricao and Patillas)

Simarouba tulae
Simaroubacea family
Traditional Uses: Anti-malaria Anti-feedant Anti-inflammatory Anti-leukemic Anti-viral

Metabolites isolated from other species of Simaroubacea family

Quassinoids
Group of highly oxygenated terpenes Responsible for its therapeutic properties Taxonomic marker

Basic skeleton of a quassinoid C-20

To expand to the phytomedicinal knowledge of native and endemic plants of Puerto Rico and to discover their chemotaxonomy. Isolate, purify and identify chemical compounds of Guaiacum officinale and Simarouba tulae leaves. Evaluate the cytotoxic activity of pure compounds of Guaiacum officinale and Simarouba tulae leaves.

Selection of the organism

Collection of the organism

Preparation of the crude extract

Purification of chemical constituents

Biological test

Plant Collection
Jayuya

Guaiacum Officinale
1.12kg de hojas
98.5 g extracto crudo

Extraction

1H

NMR Spectrum (400MHz) of crude extract in CDCL3

Alyphatic

1H

NMR Spectrum (400MHz) of CHCl3 in CDCL3

Table 1. Cytotoxicity results for Simarouba tulae leaves extracts

Plant species Extract LC50 in g/mL from the Brine Shrimp Lethality Test 26.125 % of growth inhibition on various breast cancer cell lines MCF-7 81 ZR-75-1 80

Guaiacum oficinale

Crude

Hexane

30.765

Chloroform

0.692

91

76

Ethyl Acetate

4.479

* All extracts were evaluated at a single dose of 100 g/mL. ** Results performed by Dr. Marianela Prez Torres in the UPR-MSC.

*** LC values greater than 200 g/ml are not considered cytotoxic 50

 First

collection

Place: Patillas P.R. 2008 Extractions

Table 2. Dry weight for crude extracts and solvent used for each extraction in the first collection
Plant Extract Extract dry weight
(g) 0.02 Simarouba tulae Crude Hexane Chloroform Ethyl acetate 15.0 2.33 9.21 0.90

Table 3. Cytotoxicity results for Simarouba tulae leaves extracts against Artemia Salina test
Simarouba tulae Extract Artemia Salina Test LC50 value in g/mL

Crude Hexane

2 > 200

Chloroform
Ethyl acetate

161
35

Guayacan Aqueous Layer

Extraction: Chloroform

Aqueous layer

Organic layer
Drying at Room Temp.

Guayacan aqueous layer

TLC hexane/ethyl acetate (9:1)

Rotoevaporation

Extract

chloroform/m ethanol (9.8:0.2)

TLC
Chloroform /Methanol (9.8:0.2) Hexane/Ethyl Acetate (9:1)

 Second

collection

Place: Maricao P.R. 2009 Preparation of the extracts

Table 6. Dry weight for crude extracts in the second collection of Simarouba tulae
Extract
Simarouba tulae Crude

Extract dry weight (g)

0.02 113.00

Hexane
Chloroform Ethyl Acetate

30.88
39.64 6.08

Table 7. Cytotoxicity results for Simarouba tulae leaves extracts

Artemia Salina Test
LC50 value in g/mL

Simarouba
tulae Extract

Breast Cancer Cell

Growth inhibition % *

MCF-7

ZR-75-1

T47D

Crude Hexane

23.703 > 200

82 87

< 80 < 80

94 92

Chloroform
Ethyl acetate

157.141
26.243

95
< 80

< 80
< 80

97
92

Table 7. Cytotoxicity results for Simarouba tulae leaves extracts

Simarouba Artemia Salina Test LC50 value in g/mL Breast Cancer Cell

tulae
Extract

Growth inhibition % *
MCF-7

ZR-75-1

T47D

Crude
Hexane Chloroform

23.703
> 200 157.141

82
87 95

< 80
< 80 < 80

94
92 97

Ethyl acetate

26.243

< 80

< 80

92

Table 7. Cytotoxicity results for Simarouba tulae leaves extracts

Artemia Salina Test
LC50 value in g/mL

Simarouba
tulae Extract

Breast Cancer Cell

Growth inhibition % *

MCF-7

ZR-75-1

T47D

Crude Hexane

23.703 > 200

82 87

< 80 < 80

94 92

Chloroform
Ethyl acetate

157.141
26.243

95
< 80

< 80
< 80

97
92

Chloroform extract (40g)

Chloroform extract (40g)

CC Si gel (95:5 CHCl3/MeOH)

Chloroform extract (40g)

CC Si gel (95:5 CHCl3/MeOH) TLC (9.8:0.2 CHCl3/MeOH) NMR Analysis

28 fractions

Chloroform extract (40g)

CC Si gel (95:5 CHCl3/MeOH)

28 fractions

SH2C3

CC Lipophilic Sephadex (95:5 CHCl3/MeOH)

Chloroform extract (40g)

CC Si gel (95:5 CHCl3/MeOH)

TLC (97:3) CHCl3/MeOH) CC Si gel (95:5 CHCl3/MeOH)

28 fractions

SH2C3C

SH2C3

CC Lipophilic Sephadex (95:5 CHCl3/MeOH)

Chloroform extract (40g)

CC Si gel (95:5 CHCl3/MeOH)

28 fractions

SH2C3C-C
SH2C3C

SH2C3

CC Lipophilic Sephadex (95:5 CHCl3/MeOH)

Analysis using NMR spectroscopy

Figure 4. 1H NMR Spectrum (400 MHz) of SH2C3C-C in CDCl3 Aliphatic

Allylic

Vinylic

-Heteroatoms

Analysis using NMR spectroscopy

Figure 4. 13C NMR Spectrum (100 MHz) of SH2C3C-C in CDCl3

Vinylic
Carbonyls

Oxygenated

Aliphatic

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 leaves of S. tulae
Alkenes

Oxygenated Cs C-C bonds

 Every extract of Guaiacum officinale presented activity in the Artemia Salina bioassay. From spectroscopic chloroform extracts of Guaiacum officinale are rich in metabolites.

The chloroform extract of Simarouba tulae leaves showed high cytotoxic activity against Artemia Salina and two breast cancer cell lines. From spectroscopic data SH2C3C-C is rich in metabolites The hexane extract of Simarouba tulae leaves showed high cytotoxic activity against two breast cancer cell lines.

 Identification of the main compound of the Chloroform extract using NMR spectroscopy. Perform a Chromatographic analysis of Chloroform To evaluate the cytotoxicity of pure compounds against cancer cell lines.

Batista, J.; Braz, R.; Curcino, I.; Da Silva, M.; Rodrigues E.; Vireira, P. 20(R)- and 20(S)- Simarolide Epimers 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 not Correlate 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 using Artemia 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 and Their 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 and purification of quassinoids from Quassia amara L. J. Chromato. 1984, 283, 436-440. Ospina, C. A.; Pagn, M.; Carvajal, A.; Claudio, K; Rivera, J.; Ortiz, I.; Hernndez, J. In Cytotoxic Screening of Tropical Plants Using Brine Shrimp Lethality Test.; Montes, E. L.; Eds.; Cuadernos de Investigacin Number 7; Instituto de Investigaciones Interdisciplinarias: Cayey, 2009; 1-20.

Advising

Technical Support

Claudia Ospina, PhD Mayra Pagan, PhD

Chemistry Department
Melvin De Jesus, UPR Humacao

Financial Support

Dean of Academic Affairs

Collaborators

Augusto Carvajal, M.S Marianela Prez School of Pharmacy UPR Karla Claudio- Graduate student

Undergraduate students

Ospina and Pagans research group

Janibeth Hernndez-Graduate student

Reynaldo J. Morales Rodriguez Yadira D. Cora Amaro Elsa M. Luciano Nunez Claudia A. Ospina, Ph.D. Mayra Pagn Ortiz, Ph.D.

Saline Solution

Yeast

Marine salt

Distilled water

Brine shrimp incubation Specialized recipient

Incubate @ 22-29 C for 48 hours Larvae emerges by phototropic effect

Samples Preparation
Positive Control: Berberine Chloride Negative Control: Saline Solution Plants extractions dissolved in DMSO

Bioassay
Prepare the concentration assigned to each line Add 10-15 brine shrimps by pippeting

Incubate for 24 hours

Data Collection Count the death shrimps Add MeOH Count the total shrimps