CUADERNOS DE INVESTIGACIÓN

INSTITUTO DE INVESTIGACIONES INTERDISCIPLINARIAS

UNIVERSIDAD DE PUERTO RICO EN CAYEY

Cytotoxic Screening of Tropical Plants

Using Brine Shrimp Lethality Test

Dra. Claudia A. Ospina-Millán

Dra. Mayra Pagán-Ortiz
Augusto Carvajal
Karla Claudio
Jaymie Rivera
Isamar Ortiz
Janibeth Hernández

Cuaderno 7
Año 2009
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© COM / MPO
 Cytotoxic Screening of Tropical Plants Using Brine Shrimp Lethality Test

Abstract

In recent years the study of plants has been the subject of renewed interest as a source of natural

products with medicinal value. This study aims at the development and application of bioassays

in order to detect potential sources of cytotoxic, and antitumour compounds from endemic and

native plants from Puerto Rico. For this purpose, as a strategy for primary evaluation, the brine

shrimp lethality test was chosen. The native and endemic species selected for the study were:

Canella winterana, Croton Discolor, Goetzea elegans, Guaiacum officinale, Pimenta racemosa ,

Simarouba tulae and Thouinia striata. These species were dried and extracted with a mixture of

CH2Cl2-MeOH (1:1). The crude extract was analyzed by 1H-NMR spectroscopy and afterwards

it was suspended in water and extracted with solvents of different polarities. Fifteen of the

twenty-eight extracts were active exhibited a LC50 ≤ 200 µg/mL. The most promising activity

was displayed by extracts of Simarouba tulae and Guaiacum officinale with lethality values of 2

and 4, respectively.

Key words: Brine shrimp lethality test, endemic and native plants, cytotoxic activity

Introduction

Natural products are organic compounds that are present in living organisms, animals and

plants. These are divided in two groups: primary and secondary metabolites. Primary metabolites

are those compounds which occur in all cells and play a central role in the metabolism and

reproduction of those cells. However, secondary metabolites such as polyketides, fatty acids,

terpenoids, phenylpropanoids and alkaloids are characteristic of a limited range of species and do

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not have a direct role in plant growth and development. Secondary metabolites have evolved as

defense agents against predators and the surrounding environment.1 In addition, these

compounds exert their biological effect within the organism that is responsible for their

production and can affect functions in other organisms. For example, some plant metabolites can

suppress cell division, a trait that makes them useful in treating cancer in human patients.

Moreover, these plant products also have a wide variety of different uses in health human and

industrial applications. It has been estimated that over 40% of medicines have their origins in

these secondary metabolites. 2

Due to the wide application at pharmacological level, medicinal plants have gain

importance worldwide. The work in this area begins with an assessment of the biological activity

of crude (total lipid) extracts from plants. These preliminary results provide further access for an

exhaustive analysis of its principal constituents. The lack of information related to the specific

compounds and its biological effect in plants and other organisms, provides alternative lines of

investigations, drug discoveries, and chemical modifications.

Endemic plants are species unique to a particular geographic location whereas native

plants are species belonging to a region if its presence in this region is the result of only natural

resources. Endemic and native Caribbean plants have been less studied that those from Africa,

India and Europe thus, a preliminary biological screening and subsequent isolation of the

secondary metabolites from these plants would be a great contribution to document and expand

the chemotaxonomic knowledge of these species.

During the last two decades, there have been reports of the biological screenings of the

molluscicidal (2002)3, antimycobacterial (1998 and 2001) 4,5, antiplasmodial (2001) 5, and
6,7
antibacterial (1996, 2006) activities of some tropical plants including Musaceae, Labiatae,

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Rutaceae, Myrtaceae, Apiaceae, Rubiaceae, and Malvaceae from Puerto Rico. Additionally, two

studies about the cytotoxic activity of some Puerto Rican plants has been documented. The first

was reported by Guerrero and Robledo in 1993.8 In this work, six crude extracts of the

Euphorbiaceae, Solanaceae, Myrsinaceae, Polygonaceae, and Polygalaceae families showed

LC50 values below 200 µg/mL in the Artemia Salina Test, indicating the potential presence of

bioactive compounds. No isolation of bioactive compounds was reported later. In the second

report by Chavez et al. tested the dichloromethane portion of the ethanol extract in a

concentration of 1000 µg/mL using the brine shrimp assay. 9 The extracts with a LC50 ≤ 1000

µg/mL were further tested against Hela and CHO cells. The extracts from Annona glabra,

Simarouba tulae, Tithonia diversifolia, Dendropanax arboreous, Piper jacquemontanium,

Annona montana, Polygala hecatantha were active in both assays.

These previous reports document the biological importance of secondary metabolites

from Puerto Rican plants as potential antitumour agents. The isolation and characterization of

the metabolites responsible for the biological activities is needed, as well as the study of some

other species from families that have produced bioactive compounds.

In the present study, seven species of native and endemic plants present in Puerto Rico

were studied. These were: Canella winterana, Pimenta racemosa, Guaiacum officinale, Croton

discolor, Gotzea elegans, Thouinia striata and Simarouba tulae,

Canella winterana is a tree found naturally in Florida and the Caribbean. Some important

constituents of this plant are monoterpenes like canellal (1)10, eugenol (2)11, eucalyptol (3)11, and

drimane sesquiterpenoids like 9α-hydrocinnamolide (4)12 (Figure 1). The sesquiterpenoids

exhibited phototoxic activity in a Lemna minor bioassay13. The drimane sesquiterpenoids

isolated from other species of the Canellaceae family are known for their broad antifeedant,

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antifungal, molluscicidal, and cytotoxic activities, however, the bioactivity of the Canella

winterana secondary metabolites have not been reported.

O H O H
OH O
H3C OH H3C OH
CHO OCH3 CHO

H H

canellal (1) eugenol (2) eucalyptol (3) 9-hydrocinnamolide (4)

Figure 1. Some Compounds Isolated from Canella winterana.

Pimenta racemosa is a native tree to the islands of the Caribbean, that is used in folk

medicine for the treatment of different diseases, as tooth ache, abdominal pain, fever, influenza,

rheumatism and pneumonia. This three is well known for the essential oil present in leaves and

stems. This oil is composed of eugenol (5) and terpenes, such as α-terpineol (6) among other of

its respective derivatives (Figure 2). In 2001, García et al. reported the isolation of the

triterpene lupeol (8) and the anti-inflammatory effect of the methanol extract of this species. 14

In 2004, Saenz et al reported the antibacterial and antinociceptive activity of the essential oils

and the aqueous extract of the plant. 15

OH
OCH3

OH
HO

eugenol (5) terpineol (6) lupeol (7)

Figure 2. Some Compounds Isolated from Pimenta racemosa

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 Guaiacum officinale is a tree common in the Antilles and tropical zones of America. The

bark and the resin are used to treat rheumatism, tooth ache and skin disorders. The fruits and

leaves contain triterpene saponins such as guaianin and officigenin (8).16 The resin contains

lignans such as α-guaiaconic acid (9). (Figure 3).17

O
H3CO

HO OH
COOH O
COOH
OCH3
H 3C CH3
HO
HO HO

officigenin (8) guaiaconic acid (9)

Figure 3. Some Compounds Isolated from Guaiacum officinale

Croton discolor is a shrub of the floral region of Puerto Rico and the Virgen Islands. Folk

medicine has used the tea from the leaves to treat rheumatism. Young leaves and branch tips

have been used to treat coughs. Two aporphine alkaloids, crotonosine (10) and discolorine (11)

had been isolated from this plant (Figure 4).18

HO HO

NH NCH3
H3CO H3CO

O HO
crotonosine (10) discolorine (11)

Figure 4. Alkaloids Isolated from Croton discolor

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 Simarouba tulae, is an endemic tree of Puerto Rico belonging to the Simaroubaceae

family. Some species have been used in folk medicine as fever reducer, tonic and antimalarial. In

the last years, its possible therapeutic purposes have increased because of its antimalarial, anti-

inflammatory, antileukemic, antifeedant and antiviral activities. 19 In 1997, Chavez reported the

cytotoxic activity of Simarouba tulae against Hela and CHO cells.9 No studies about the chemical

constituents of this species had been reported.

Finally, Thouinia striata (Sapindaceae) and Gotzea elegans (Solanaceae) are also

endemic plants of Puerto Rico. To our knowledge, no references about the secondary metabolites

and biological activity of these species had been reported.

An efficient, rapid, and general test for evaluate the cytotoxic activity of extracts and

compounds from plants is the brine shrimp lethality bioassay. This bioassay has a good

correlation with cytotoxic activity in some human solid tumors and pesticide activity. 20,21 We

decided to study the cytotoxic activity of selected tropical plants from Puerto Rico using the

brine shrimp lethality test, and in the future use a bioassay-guide fractionation to guide the

purification, isolation and subsequent characterization of the active constituents.

Materials and Methods

General Experimental Procedures

The NMR spectra were recorded on an Anasazi 60 MHz FT-NMR or in a Bruker (400

MHz). The chemical shifts are given in δ (ppm). Samples were dissolved in CDCl3 or (CD3)2SO

and tetramethyl silane (TMS) was used as internal reference. The alive shrimp in the Artemia

salina bioassay were counted by inspection of the well with the aid of a Stereo zoom microscope

(10X widefield eyepieces 45 degree inclined body, 7X to 45X magnification). Silica Gel (70-

230 mesh, 60A) was used for column chromatography. The deuterated solvents (CDCl3 and

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(CD3)2SO), TMS, berberine chloride, hexane, dichloromethane and methanol were purchased

from Sigma Aldrich. Celite (521), chloroform and ethyl acetate were purchased from Thermo

Fisher Scientific. Brine shrimp eggs were acquired in a local pet shop (San Juan, Puerto Rico)

and the yeast was purchased in a local grocery store (Caguas, Puerto Rico).

Plant Material

The leaves of Canella winterana, Thouinia striata, Croton discolor and Guaiacum

officinale were collected at the dry forest in Guánica, PR in February 2008. The leaves of

Pimenta racemosa were collected in Cidra, PR in February 2008. The leaves of Goetzea elegans

were colleted at the Botanical Garden of the University of Puerto Rico in Río Piedras. The

leaves of Simarouba tulae were collected in Patillas, PR. The plants were identified by professor

Augusto Carvajal of the Department of Biology at the University of Puerto Rico at Cayey. A

voucher specimen was reserved to be deposited in the herbarium of the Botanical Garden of the

University of Puerto Rico.

Extraction and Isolation

The air-dried leaves of Canella winterana (283.80 g) were crushed in a blender with

three portions of 1L of CH2Cl2/CH3OH (1:1, v/v). The solid debris was removed from the

combined extracts by a Celite filtration and the extract was concentrated under vacuum to yield

65.09 g of crude. The extract was suspended in 500 mL of water and extracted with solvents of

increasing polarity, hexane (3 x 150 mL), chloroform (3 x 200 mL) and ethyl acetate (3 x 150

mL). Each of the extracts was concentrated to dryness by rotoevaporation. The weights of the

extracts were hexane 25.01 g, chloroform 15.76 g and ethyl acetate 2.10 g.

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 The air-dried leaves of Croton discolor (46.59 g) were crushed in a blender with three

portions of 500 mL of CH2Cl2/CH3OH (1:1, v/v). The solid debris was removed from the

combined extracts by a Celite filtration and the extract was concentrated under vacuum to yield

8.50 g of crude. The extract was suspended in 300 mL of water and extracted with solvents of

increasing polarity, hexane (3 x 100 mL), chloroform (3 x 100 mL) and ethyl acetate (3 x 100

mL). Each of the extracts was concentrated to dryness by rotoevaporation. The weights of the

extracts were hexane 0.75 g, chloroform 3.09 g and ethyl acetate 0.19 g.

The air-dried leaves of Goetzea elegans (270.11 g) were crushed in a blender with three

portions of 1L of CH2Cl2/CH3OH (1:1, v/v). The solid debris was removed from the combined

extracts by a Celite filtration and the extract was concentrated under vacuum to yield 25.10 g of

crude. The extract was suspended in 500 mL of water and extracted with solvents of increasing

polarity, hexane (3 x 150 mL), chloroform (3 x 150 mL) and ethyl acetate (3 x 100 mL). After

removal of the solvents, the weights of the dry extracts were hexane 5.88 g, chloroform 1.06 g

and ethyl acetate 0.52 g.

The air-dried leaves of Guaiacum officinale (270.14 g) were crushed in a blender with

three portions of 1L of CH2Cl2/CH3OH (1:1, v/v). The solid debris was removed from the

combined extracts by a Celite filtration and the extract was concentrated under vacuum to yield

44.61 g of crude. The extract was suspended in 500 mL of water and extracted with solvents of

increasing polarity, hexane (4 x 200 mL), chloroform (4 x 200 mL) and ethyl acetate (3 x 50

mL). After removal of the solvents, the weights of the dry extracts were hexane 5.05 g,

chloroform 4.96 g and ethyl acetate 2.06 g.

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 The air-dried leaves of Pimenta racemosa (279.66 g) were crushed in a blender with

three portions of 1L of CH2Cl2/CH3OH (1:1, v/v). The solid debris was removed from the

combined extracts by a Celite filtration and the extract was concentrated in vacuum to yield

17.16 g of crude. The extract was suspended in 500 mL of water and extracted with solvents of

increasing polarity, hexane (3 x 450 mL), chloroform (3 x 100 mL) and ethyl acetate (3 x 100

mL). After removal of the solvents, the weights of the dry extracts were hexane 7.74 g,

chloroform 1.00 g and ethyl acetate 6.52 g.

The air-dried leaves of Simarouba tulae (65.71 g) were crushed in a blender with three

portions of 500 mL of CH2Cl2/CH3OH (1:1, v/v). The solid debris was removed from the

combined extracts by a Celite filtration and the extract was concentrated under vacuum to yield

15.03 g of crude. The extract was suspended in 400 mL of water and extracted with solvents of

increasing polarity, hexane (3 x 100 mL), chloroform (3 x 100 mL) and ethyl acetate (3x 100

mL). After removal of the solvents, the weights of the dry extracts were hexane 2.33 g,

chloroform 9.21 g and ethyl acetate 0.90 g.

The air-dried leaves of Thouinia striata (106.39 g) were crushed in a blender with three

portions of 1L of CH2Cl2/CH3OH (1:1, v/v). The solid debris was removed from the combined

extracts by a Celite filtration and the extract was concentrated under vacuum to yield 15.84 g of

crude. The extract was suspended in 500 mL of water and extracted with solvents of increasing

polarity, hexane (3 x 150 mL), dichloromethane (3 x 100 mL) and ethyl acetate (3 x 100 mL).

Each of the extracts was concentrated to dryness by rotoevaporation. The weights of the extracts

were hexane 3.61 g, dichloromethane 6.03 g and ethyl acetate 0.13 g.

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Brine shrimp lethality bioassay

Brine shrimp lethality bioassay was performed to assess the cytotoxicity of the crude and

extracts of the plants.20 The bioassay was performed as reported earlier with some minor

modifications. Brine shrimp eggs were hatched in artificial seawater (0.5 g eggs per liter) at the

dark portion of a divided chamber. The artificial seawater was prepared using sea salt 30 g/L,

containing 0.006 g of yeast as food source. After approximately 48 h the phototropic nauplii

move through a hole in the division to the portion of the camber kept under continuous light.

The nauplii were collected with a pipette and concentrated in a beaker. The concentration of the

nauplii was adjusted adding seawater to the beaker until approximately 10-15 nauplii were found

in 100 µL of solution measured with an automatic micropipette.

The sample (0.002g) to be tested was dissolved in 100 µL of DMSO and diluted with

seawater (1900 µL) to a concentration of 1 mg/mL. Berberine chloride was used as a positive

control and prepared according to the sample. The blank solution (negative control) was prepared

diluting 50 µL of DMSO in 950 µL of seawater. The bioassay was conducted in a 96 microwell

plate. Several solutions of each sample ranging from 1.95 µg/mL to 500 µg/mL were prepared

in triplicate. To each solution 100 µL of seawater containing 10-15 nauplii were added. The

microwell plate was incubated at room temperature during 24 h. with constant lighting.

After 24 h the dead nauplii were counted with the aid of a microscope and the lethal

concentration (LC50 value) was calculated by probit analysis. Larvae were considered dead if

they did not move during the observation. The LC50 value was obtained by regression analysis of

the data of percentage lethality versus concentration.

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Results and Discussion

The selection of the plant species for this project was based primarily on the little

information regarding their chemical composition in spite of the fact that some of the species

belong to families well recognized for the bioactivity of its compounds. In addition, most of the

selected species are native, meaning that they occur naturally in a region (the Caribbean) and

two of them are endemic species, meaning that they are prevalent in a particular locality (the

island of Puerto Rico). Two important aspects of this project are worth to be mentioned, first

the importance of identifying secondary metabolites that may be unique to endemic species. The

identification of such chemotaxonomic markers may aid in the identification of species by its

chemical composition and it demonstrates the multidisciplinary character of the project. In

addition, the isolation and characterization is guided by the bioactivity found in the extracts of

the species.

The solid-liquid extraction that occurs in the blender with dichloromethane/methanol

produces a crude mixture of secondary metabolites. The complex mixture is further simplified

by liquid-liquid extraction suspending the crude in water and using solvents of increasing

polarity to separate each class of metabolites based on its polarity. The crude extract of each

species was analyzed by 1H-NMR Spectroscopy and its response to the Artemia salina bioassay

was assessed. The extracts of the crude were also screened by the Artemia salina bioassay.

The most difficult challenge in natural products chemistry is the separation and

purification of a compound from a complex mixture and its further identification. NMR

spectroscopy is the most important tool for structure elucidation and it is extensively used for

that purpose. However, when working with complex mixtures the interpretation of the spectra is

difficult because of the crowding and overlapping of signals. The 1H-NMR spectra of the total

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lipid extraction shows the occurrence of functional groups according to the chemical shift of the

protons. The area of the signal is proportional to the abundance of that type of proton in the

mixture. The absence or low intensity of any signal does not necessarily indicate the absence of

the corresponding proton, because the signal may be diminished by strong signals in the

spectrum. The results form the 1H-NMR spectra for the total extract is summarized in Table 1

and individual spectrum are shown in Figures 5-10.

All of the species show absorbance in the region corresponding to aliphatic and allylic

protons being one of these signals the predominant in the spectrum of Canella winterana,

Thouinia striata, Guaiacum officinale, and Goetzea elegans. Pimenta racemosa has the most

predominant signal in the aromatic region. In the spectrum of Pimenta racemosa the strong

signals in 3.5 and 4.4 ppm correspond to methanol and water respectively. In the spectrum of

Guaiacum officinale the signal at 3.5 ppm correspond also to residual methanol from the

extraction.

The occurrence of most of the proton types in Canella winterana is consistent with the

variedty of functional groups as seen in Figure 1. In the Goetzea elegans spectrum the most

predominant signals correspond to aliphatic protons although there are minor resonances in the

vynilic and allylic regions of the spectrum. In Pimenta racemosa there is evidence in the

spectrum of the occurrence in the species of aromatic and olefinic functional groups

corresponding to compounds such as eugenol and its derivatives (Figure 2). The spectrum of

Simarouba tulae has signals across the different chemical shifts. It is worth mentioning that the

region from 3.0 to 4.8 has several signals corresponding to protons adjacent to heteroatoms,

which could be due to the presence of highly oxygenated species as the quassinoids. The

aliphatic protons are dominant in the 1H- NMR spectrum of Goetzea elegans with minor signals

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in the aromatic, vynilic and α-heteroatom regions. Thouinia striata has its principal proton

signals in the aliphatic and allylic regions. None of these last two species has literature precedent

regarding their chemical composition.

Table 1. Types of Proton in the 1H-NMR of the Total Extract

Allylic α-Heteroatoms Vinylic Aromatic
Aliphatic
Species X=C-CH3 H-C-Y C=C-H Ar-H
(0.2-1.5)
(1.5-3.0) (3-4) (4.5-5.5) (6.5-8.0)
Canella winterana √ √ √ √ √
Thouinia striata √ √ √
Guaiacum officinale √ √ √ √
Pimenta racemosa √ √ √ √ √
Goetzea elegans √ √ √ √
Simarouba tulae √ √ √ √
X= C, O, N
Y= X, O, N, S

Figure 5. 1H NMR spectrum (60 MHz) of Canella Winterana in CDCl3

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 Figure 6. 1H NMR spectrum (60 MHz) of Thouinia striata in CDCl3

Figure 7. 1H NMR spectrum (60 MHz) of Guaiacum officinale in CDCl3

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 Figure 8. 1H NMR spectrum (60 MHz) of Pimenta racemosa in CD3OD

Figure 9. 1H NMR spectrum (60 MHz) of Goetzea elegans in CDCl3

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 Figure 10. 1H NMR spectrum (400 MHz) in CDCl3

Seven plants were collected, dried and extracted with a mixture of CH2Cl2-MeOH (1:1).

The resulting crude extract was suspended in water and extracted with solvents of different

polarities. We used the bioassay-guided fractionation and tested the extracts for the cytotoxic

activity using the brine shrimp lethality test. Table 2 show that 15 extracts of 6 species exhibited

LC50 values below 200 µg/mL. The most promising activity was displayed by crude extracts of

Simarouba tulae, Guaiacum officinale and Croton discolor with lethality values of 2, 21 and 111

µg/mL, respectively. These species belong to families of plants whose genera have demonstrated

contain compounds with anticancer activity such as quassinoids, lignans and cembranes. In

addition the crude extracts also show activity, which infers that the cytotoxic activity can be

attributed to a particular class of compounds. In contrast, the crude extracts of Canella, Pimenta

and Gotzea elegans did not show activity against Artemia salina, but some extracts resulting

from solvent extraction showed activity. This may be due to antagonistic effects of the complex

mixture of compounds present in the crude extract.

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 From this preliminary screening we identified extracts of Puerto Rican plants with

cytotoxic activity. Subsequent isolation and identification of the active constituents is needed, as

well as the determination of other possible bioactivities as antimicrobial and the testing against

specific cancer cell lines.

Table 2. Brine Shrimp Lethality Data of Puerto Rican Plants

Plant Extract LC50 value in µg/mL

Canella winterana Crude >200
Hexane 78
Chloroform >200
Ethyl Acetate >200
Croton discolor Crude 111
Hexane 132
Chloroform >200
Ethyl Acetate >200
Gotzea elegans Crude >200
Hexane 91
Chloroform 188
Ethyl Acetate >200
Guaiacum officinale Crude 21
Hexane 87
Chloroform 3
Ethyl Acetate 4
Pimenta racemosa Crude >200
Hexane 47
Chloroform 86
Ethyl Acetate 189
Simarouba tulae Crude 2
Hexane >200
Chloroform 161
Ethyl Acetate 35
Thouinia striata Crude >200
Hexane >200
Dichloromethane >200
Ethyl Acetate >200

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Aknowledgements

This work was supported by Institutional funds provided by the “Instituto de

Investigaciones Interdisciplinarias” at UPR-Cayey. Jaymie Rivera Gutierrez and Isamar Ortiz

Rivera thanks the Amgen Bio-Minds Program and the PR-LSAMP Program for their financial

support. We are grateful to personnel of the Chemistry and Biology Departments for the

technical assistance. We also acknowledge Melvin De Jesus and the NMR facilities from the

Department of Chemistry at UPR-Humacao.

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activity of essential oils of Pimenta racemosa var. terebinthina and Pimenta racemosa var.
grisea” Fitoterapia, 2004, 75, 599-602.

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16
Ahmad, V. U.; Bano, N.; Bano, S. “Officigenin, a New Sapogenin of Guaiacum officinale” J.
Nat. Prod., 1984, 47, 977–982.

17
Little, E. L. Jr.; Wadsworth F. H.; Common Trees of Puerto Rico and the Virgin Islands, Ag.
Handbook 249, USDA, Washington D. C. 1964.

18
Stuart, K. L.; Cava, M. P. “Proaporphine alkaloids” Chem. Rev., 1968, 68, 321-339.

19
Engler, A.; Prantl, K. “Simaroubaceae” Mart F1 Bras. 1872, 12, 198-202.

20
Meyer, B. N.; Ferrigni, N. R.; Putnam, J. E.; Jacobsen, L. B.; Nichols, D. E.; McLaughlin J. L.
“Brine Shrimp: A Convenient General Bioassay for Active Plant Constituents” Planta Médica
1982, 45, 31-34.

21
Sam, T. W. “Toxicity Testing Using the Brine Shrimp: Artemia Salina. Colegate, S. M. and

Molyneux, R. J. Eds. Bioactive Natural Products Detection, Isolation, and Structural

Determination. CRC Press, Boca Ratón, FL. 1993, 442-456.

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 Canella winterana
Classification

Kingdom Plantae
Canella winterana it Subkingdom Tracheobionta
is also known as Superdivision Spermatophyta
canella alba, wild Division Magnoliophyta
cinnamon and Class Magnoliopsida

barbasco Subclass Magnoliidae

Order Magnoliales
Family Canellaceae
Genus Canella
General Information Species Winterana
While the outer bark is gray the inner
Canella winterana, also known as wild
bark is yellow and aromatic with the
cinnamon is a salt tolerant evergreen
smell of mixed spices. The generic name is reputed
shrub. It has an open canopy that
to originate from the Latin
reaches a height of 10 meters with a Distribution
diameter of 8 inches. Purple and white This species occurs naturally from south word for cinnamon as it is
showy flowers cover the tree in summer Florida through the West Indies and used in Spanish “canela”.
and fall followed by bright red berries has been introduced in Brazil and
The species epithet honors a
clustered near the tips of the branches. Venezuela.
captain Winter who first
Traditional Uses introduced its bark to
sesquiterpenes, and warburganal
In folk medicine, the inner bark tea is Europe.
among other non identified
used to treat fevers, relieve indigestions
compounds.
and is gargled to treat inflamed tonsils.
Externally, the leaves are applied to Canellal, isolated from bark is

relieve rheumatism and headache. antifungal, antimicrobial, cytotoxic,

The rum extract of the bark is used as a and inhibits insect feeding.

liniment to relieve rheumatism and The bark and leaves are still used in
other pains, and its liquor is consumed the West Indies to spice beverages
to treat stomach pains. and season food.

On distillation the bark yields a volatile The volatile oils found in the leaves
oil containing benzoyleugenol, have also been used as additives in
caryophyllene resins, canellal, cineol, perfumes.
clovanidiol, helicid, mannitol,
myristicin, 1-pinene, drimane

1) Plants Profile for Canella winterana http://plants.usda.gov/java/profile?symbol=CAWI accessed June 2009

2) Canella winterana hort.ufl.edu/shrubs/CANWINA.PDF accessed June 2009
3) Nellis, D. W. Poisonous Plants and Animals of Florida and the Caribbean 1st Ed. Pineapple Press Inc, 1997

© MPO/COM
 Croton discolor
Classification

Kingdom Plantae
Subkingdom Tracheobionta
Superdivision Spermatophyta
This plant is also called
white maran. Division Magnoliophyta
Class Magnoliopsida
Subclass Rosidae
Order Euphorbiales
Family Euphorbiaceae
Genus Croton L.
Species Discolor
General Information Variety willd (lechecillo)

Croton discolor is an aromatic 8 ft. tall methylcrotonosine, linearisine and

shrub. It has alternate leaves which
are dark on the top lighter on the
undersides. It has male an female
flowers which born on separate stalks.
Its seeds are 0.25 inches long.
The plant toxicity is attributed to the
alkaloids crotonosine,
dihydrosalutadirine,
discolorine, which have been isolated The species name is Latin
from the plant. and it refers to the fact that
the top and the bottom of
Distribution its leaves are of different
color.
The genus of over 600 species of is
distributed worldwide and includes
8,14-
aromatic herbs, shrubs and trees.

Traditional Uses
Folk medicine has used the tea from Externally, the oil is an irritant and
the leaves to treat rheumatism. Young may cause blistering of the skin
leaves and branch tips have been The esters of theteracyclic
used to treat coughs. A component of diterpenoid phorbol have been
croton oil (phorbol 12-tiglate 13 found to promote tumors in mouse
decanoate) has been found to inhibit skin previously treated with
leukemia. Croton oil found in leaves, subcarcinogenic doses of
stem and seeds is effective in small carcinogens.
doses but in high doses causes severe
Croton discolor from Isla de Mona
gastroenteritis. Puerto Rico

1) Plant Profile for Croton discolor USDA http://plants.usda.gov/gallery/thumbs/crdi8_001_tvp.jpg accessed June 2009
2) Nellis, D. W. Poisonous Plants and Animals of Florida and the Caribbean 1st Ed. Pineapple Press Inc, 1997

©MPO/COM
 Goetzea elegans
Classification

Kingdom Plantae
Goetzea elegans Subkingdom Tracheobionta
is an endemic
Superdivision Spermatophyta
plant of Puerto
Division Magnoliophyta
Rico also known
Class Magnoliopsida
as beautiful,
Matabuey and Subclass Asteridae
Manzanilla. Order Solanales
Family Solanaceae
Genus Goetzea Wydler.

Species Goetzea elegans

General Information
Goetzea elegans is characterized by its
trumpet shaped orange flowers. It is an
evergreen shrubby tree that can grow
up to 9 meters in height. This endemic
species is only found in the semi-
evergreen forests in northern Puerto
Rico. In 2005 fewer than fifty individuals
of the species remained. It is very rare,
it can be found in moist limestone and
moist coastal forests. Goetzea elegans
pertains to the Solanaceae family also
Wydler
known as the potato or nightshade family
that has 75 genera and over 3000
species. Several species of this family
have provided many poisons as atropine
and scopolamine that can be deadly in
large amounts.
Several vegetables of the Solanaceae
family are important for their nutrient
components these include the tomato,
eggplant, potato and chili pepper.

Solanaceae family
In the solanaceae family the leaves
can be opposite, clustered or
alternate. There is also variety in the
Atropine has been isolated from
arrangement and the form of the
several species of the Solanaceae The Solanaceae family
flowers, but generally the corolla is
family. It is a secondary metabolite includes the potatoes,
radially symmetrical and five lobed
that serves as a drug with a wide tomatoes, peppers,
with five stamens. The calyx and
variety of effects. It is a competitive
tobacco and petunias.
corolla can be either bell-shaped,
wheel-shaped or cylinder-shaped. antagonist for the muscarinic
acetylcholine receptor.
1) Liogier, A. H.; Liogier, H. A.; Martorell, L. F. Flora of Puerto Rico and adjacent islands 2nd Ed. UPR Editorial, 2000.
2) National Collection of Imperiled Plants http://www.centerforplantconservation.org/ASP/CPC_ViewProfile.asp?CPCNum=2041 accessed
June 2009
3) Plants Profile for Goetzea elegans http://plants.usda.gov/java/profile?symbol=GOEL accessed June 2009
4) Kirkpatrick, Z. M. Wildflowers of the western plains 1st Ed. University of Texas Press, 1992

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 Guaiacum officinale
Classification

Kingdom Plantae

Guaiacum officinale is Subkingdom Tracheobionta

also known as the tree Superdivision Spermatophyta

of life, it is the source of Division Magnoliophyta
the true lignum vitae, a Class Magnoliopsida
trade wood also called Subclass Rosidae
guayacan. Order Sapindales
Family Zygophylaceae
Genus Guaiacum L.
Species Guaicaum
General Information heated, when it emits an agreeable officinale L
Guaiacum officinale is an ornamental scent. This wood was once very important

evergreen tree with pretty rich blue for uses requiring strength, weight, and

flowers, the trunk is a greenish-brown hardness. All species of the genus are
COOR2
color, the wood of slow growth but now listed in CITES (Convention on
attains a height of 40 to 60 feet, stem International Trade in Endangered R1O

almost always crooked, bark furrowed; Species of Wild Fauna and Flora) as
R1= azúcares
the wood is extraordinarily heavy, solid potentially endangered species. The bark
R2= H o azucares
and dense, fibers cross-grained; yields 1 per cent volatile oil of delicious
Saponins form G.
pinnate leaves, oval obtuse; fruit fragrance. officinale
obcordate capsule; seeds solitary,
Distribution
hard, oblong. The wood has a slight
This species occurs naturally in Florida
acrid taste and is odorless, unless
Puerto Rico and the Virgin Islands.
Traditional Uses
Guaiacum resin is an acrid stimulant. It It is believed that the guaiacum did not
has been used to treat rheumatism, had power to cure the syphilis but
chronic skin diseases, scrofula and to rather alleviate the symptoms.
prevent gout. As a remedy for
A number of saponins have been
venereal diseases guaiacum wood
reported from different plant parts.
was successfully used. Nine years after
Guaicins A to G have been found in
its introduction to Europe more than
leaves, stem bark and fruits.
Guaiacum officinale is
three thousand persons had benefit
Heartwood contains lignans such as the national flower of
from its therapy Jamaica.
furoguaiacidin and furoguaiaodin.

1) Pereira, J. The Elements of Materia Medica and Therapeutics 4th Edition, Longman, 1857.
2) A Modern Herbal/Guaiacum http://www.botanical.com/botanical/mgmh/g/guaiac42.html accessed June 2009 accessed June 2009
3) Magical Flower http://www.flickr.com/photos/kam74/2462585674/ accessed June 2009
4) Plants Profile for guaiacum officinale http://plants.usda.gov/java/profile?symbol=GUOF accessed June 2009
5) Daniel, M. Medicinal Plants Science Publishers 2005.

©MPO/COM
 Pimenta racemosa
Classification
Pimenta racemosa is
also known as Kingdom Plantae
malagueta, guayabita, Subkingdom Tracheobionta
wild cinnamon and bay Superdivision Spermatophyta
rum tree. Division Magnoliophyta
Class Magnoliopsida
Subclass Rosidae
Order Myrtales
Family Myrtaceae

General Information Genus Pimenta Lindl.

grisea with its oil rich in geraniol, methyl
Pimenta racemosa (Bay rum tree) is Species Pimenta
eugenol and isoeugenol. The citriodora
native to Puerto Rico and perhaps to the racemosa
variety has strongly lemon-scented oil
Virgin Islands and the other West Indian
consisting mostly of geranial. A believed
islands. The tree is identified by its
chemovariety produces an anise-
opposite, smooth-margined, dark green
scented oil consisting mostly of methyl
leaves, which when crushed have a
chavicol and methyl eugenol.
strong bay rum (clove-cinnamon)
Harvesting and extraction of any variety
fragrance. Its bark is gray to light brown, Eugenol is the main
with true bay rum produces an oil of
white flowers borne in round clusters and constituent of bay leaf oil
inferior quality.
the fruits are small, elongated green fruits
that turn black in maturity. One of the Distribution
varieties that have been described is the Pimenta racemosa can be found in
Traditional Uses Puerto Rico and the Virgin Islands.
The leaves have been used as a spice in
cooking, as a perfume, and as a liniment
Bay Leaf Oil
Bay leaf oil or myrcia oil is yellow to dark
to ease muscle aches, P. racemosa var.
brown. The crude oil has a sweet
terebinthina is used in the Caribbean
penetrating odor. Approximately 80
basin for different afflictions. In the
compounds have been detected
Dominican Republic, the essential oil
including monoterpenes, sesquiterpenes,
extracted from the leaves is used for the
aromatic and aliphatic compounds.
treatment of rheumatism or for
The major use for bay oil is in hair lotions
toothache. P. racemosa var. grisea is
with minor uses in perfumery and as a
used for its anti-inflammatory and
food flavoring.
analgesic properties.

1) E. A. Weiss Spice Crops CABI Publishing, 2002

2) Plants Profile Pimenta racemosa http://plants.usda.gov/java/profile?symbol=PIRA accessed June 2009
3) Kirk, T. K. Tropical Trees of Florida and the Virgin Islands Pineapple Press Inc, 2009.

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 Simarouba tulae
Classification

Kingdom Plantae
Subkingdom Tracheobionta
Simarouba tulae is Superdivision Spermatophyta
an endemic plant Division Magnoliophyta
of Puerto Rico
Class Magnoliopsida
commonly known
Subclass Rosidae
as “aceitillo falso”
Order Sapindales
Family Simaroubaceae
Genus Simarouba Aubl.

Species Simarouba
tulae Urb

Simaroubaceae Family Traditional Uses

This family consists of approximately 25 The bark, wood and leaves from
genera and 150 species that are known the genus Simarouba have been
In the early twentieth
by their chemical composition that used for their analgesic, century quassinoids were
includes oxygenated triterpenes such as antimicrobial, antimalarial, introduced in Europe as
the quassinoids. Usually their trees are astringent, sudorific, nicotine substituent.
slender, with long pinnate leaves on the antidysenteric, amoebicidic and
top of a slender hole and racemes of antibacterial properties. Its bark
little berries. has been used as a remedy for the
treatment of diarrhea, dysentery
and dyspeptic affections.
O O
Quassinoids
There are about 100 quassinoids
The main active compounds of
known and some like isobruceine A
Simarouba are the quassinoids, which Basic skeleton of a
posses a strong antileukemic activity. Quassinoid C-20
belong to the triterpene chemical
Some other quassinoids have antiviral,
family and which are responsible for its
antimalarial, antifeedant and
bioactivity and are considered
insecticidal properties.
chemical markers of the family.

1) Plants Profile for Simarouba tulae http://plants.usda.gov/java/profile?symbol=SITU accessed June 2009

2) Savory, J. A compendium of domestic medicine 7th Edition 1865.
3) Daniel, M. Medicinal Plants Science Publishers 2005.
4) Wiart, C. Medicinal Plants of Asia and the Pacific CRC Press, 2006.
5) Memory, P. F. Medicinal botany 2nd Ed. John Wiley and Sons 2003.

©MPO/COM
 Thouinia striata
Classification

Kingdom Plantae
Thouinia striata is an Subkingdom Tracheobionta
endemic plant found
Division Magnoliophyta
only in the island of
Class Magnoliopsida
Puerto Rico
Subclass Rosidae
Order Sapindales
Family Sapindaceae
Genus Thouinia
Species striata
Variety portoricensis
General Information
striata
Thouinia striata is also known as dicotyledons. Thouinia striata pertains
ceboruquillo and serrasuela. This tree to the Sapindaceae botanical family,
and shrub is an endemic plant of otherwise known as the Soapberry
Puerto Rico. Ceboruquillo is part of the family. “The common name of
Magnoliophyta division otherwise the genus refers to the
known as flowering plants. This plant is
presence of saponins in
also a member of the magnoliopsida
the soft pulps of the
class usually referred to as
trees fruits. Solutions
made from this pulp

Sapindaceae family produce lather very

much like the one
Sapindaceae, also known as the Many are lactiferous, i.e. they contain
produced by
soapberry family, is a family of milky sap, and many contain mildly
manufactured soap.
flowering plants in the order toxic saponins with soap-like qualities in
Some species of the
Sapindales. There are about 140-150 the foliage and/or the seeds, or roots.
Some of the sapindaceae species are
family have been used
genera with 1400-2000 species,
including maple, horse chestnut and of economic importance including as detergents in tropical
lychee. Sapindaceae members occur edible fruits, medicinal plants, fish regions.”
in temperate to tropical regions poisons, soaps and stimulating

throughout the world. beverages.

1) ITIS Standard Report Page Thouinia striata

http://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&search_value=28708 accessed June 2009
2) Simpson, M. G. Plant Systematics Academic Press 2006
3) Nelson, G. The trees of Florida Pineapple Press Inc. 2004.

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