Reynaldo J. Morales Rodriguez

Yadira D. Cora Amaro

Elsa M. Luciano Nunez

Claudia A. Ospina, Ph.D.

Mayra Pagán Ortiz, Ph.D.

Family: Zygophyllaceae

Genus: Guaiacum

Species: Officinale

Common name: Guayacán, 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.

Family: Simaroubaceae

Commonly Known as “aceitillo falso”

Trees of 2-8 meters

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

Simarouba tulae

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

Biological test Purification of

chemical constituents

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

* 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 cytotoxic

Plant species Extract LC50 in

µg/mL from

the Brine

Shrimp

Lethality

Test

% of growth inhibition‡ on

various breast cancer cell

lines

MCF-7 ZR-75-1

Guaiacum

oficinale

Crude 26.125 81 80

Hexane 30.765 - -

Chloroform 0.692 91 76

Ethyl Acetate 4.479 - -

Table 1. Cytotoxicity results for Simarouba tulae leaves extracts

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 15.0

Hexane 2.33

Chloroform 9.21

Ethyl acetate 0.90

Table 3. Cytotoxicity results for Simarouba tulae leaves extracts against Artemia Salina test

Simarouba tulae

Extract

Artemia Salina Test

LC50 value in µg/mLᶱ

Crude 2

Hexane > 200

Chloroform 161

Ethyl acetate 35

Guayacan Aqueous Layer

Extraction: •Chloroform

Aqueous layer

Organic layer

Rotoevaporation TLC Drying at

Room Temp.

hexane/ethyl acetate (9:1)

chloroform/methanol (9.8:0.2)

Extract

Guayacan aqueous layer

• Chloroform /Methanol (9.8:0.2)

• Hexane/Ethyl Acetate (9:1)

TLC

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

Extract dry weight (g)

±0.02

Crude 113.00

Hexane 30.88

Chloroform 39.64

Ethyl Acetate 6.08

Table 7. Cytotoxicity results for Simarouba tulae leaves extracts

Simarouba

tulae

Extract

Artemia

Salina Test

LC50 value in

µg/mLᶱ

Breast Cancer Cell

Growth inhibition % *ᵜ

MCF-7

ZR-75-1

T47D

Crude 23.703 82 < 80 94

Hexane > 200 87 < 80 92

Chloroform 157.141 95 < 80 97

Ethyl acetate 26.243 < 80 < 80 92

Table 7. Cytotoxicity results for Simarouba tulae leaves extracts

Simarouba

tulae

Extract

Artemia

Salina Test

LC50 value in

µg/mLᶱ

Breast Cancer Cell

Growth inhibition % *ᵜ

MCF-7

ZR-75-1

T47D

Crude 23.703 82 < 80 94

Hexane > 200 87 < 80 92

Chloroform 157.141 95 < 80 97

Ethyl acetate 26.243 < 80 < 80 92

Table 7. Cytotoxicity results for Simarouba tulae leaves extracts

Simarouba

tulae

Extract

Artemia

Salina Test

LC50 value in

µg/mLᶱ

Breast Cancer Cell

Growth inhibition % *ᵜ

MCF-7

ZR-75-1

T47D

Crude 23.703 82 < 80 94

Hexane > 200 87 < 80 92

Chloroform 157.141 95 < 80 97

Ethyl acetate 26.243 < 80 < 80 92

Chloroform extract (40g)

Chloroform extract (40g)

CC Si gel (95:5 CHCl3/MeOH)

Chloroform extract (40g)

CC Si gel (95:5 CHCl3/MeOH)

28 fractions

NMR Analysis TLC

(9.8:0.2 CHCl3/MeOH)

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)

28 fractions

SH2C3 CC Lipophilic Sephadex

(95:5 CHCl3/MeOH) SH2C3C

TLC (97:3) CHCl3/MeOH)

CC Si gel (95:5 CHCl3/MeOH)

Chloroform extract (40g)

CC Si gel (95:5 CHCl3/MeOH)

28 fractions

SH2C3 CC Lipophilic Sephadex

(95:5 CHCl3/MeOH) SH2C3C

SH2C3C-C

Aliphatic Allylic

Analysis using NMR spectroscopy

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

α-Heteroatoms Vinylic

Analysis using NMR spectroscopy

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

Aliphatic Oxygenated Vinylic

Carbonyls

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 C’s

C-C σ 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.; Pagán, M.; Carvajal, A.; Claudio, K; Rivera, J.; Ortiz, I.; Hernández, J. In “Cytotoxic Screening of Tropical Plants Using Brine Shrimp Lethality Test”.; Montes, E. L.; Eds.; Cuadernos de Investigación Number 7; Instituto de Investigaciones Interdisciplinarias: Cayey, 2009; 1-20.

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 Pharmacy UPR

Karla Claudio- Graduate student

Janibeth Hernández-Graduate student

Reynaldo J. Morales Rodriguez

Yadira D. Cora Amaro

Elsa M. Luciano Nunez

Claudia A. Ospina, Ph.D.

Mayra Pagán Ortiz, Ph.D.

Data Collection

Count the death shrimps Add MeOH Count the total shrimps

Bioassay

Prepare the concentration assigned to each line

Add 10-15 brine shrimps by pippeting

Incubate for 24 hours

Samples Preparation

Positive Control: Berberine Chloride

Negative Control: Saline Solution

Plants extractions dissolved in DMSO

Brine shrimp incubation

Specialized recipient Incubate @ 22-29 ◦C for 48

hours Larvae emerges by phototropic effect

Saline Solution

Yeast Marine salt Distilled water