CUADERNOS DE INVESTIGACIN

INSTITUTO DE INVESTIGACIONES INTERDISCIPLINARIAS UNIVERSIDAD DE PUERTO RICO EN CAYEY

Cytotoxic Screening of Tropical Plants Using Brine Shrimp Lethality Test

Dra. Claudia A. Ospina-Milln Dra. Mayra Pagn-Ortiz Augusto Carvajal Karla Claudio Jaymie Rivera Isamar Ortiz Janibeth Hernndez

Cuaderno 7 Ao 2009

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Diseo de Portada: Prof. Harry Hernndez Encargado de la serie de cuadernos: Dr. Errol L. Montes Pizarro Directora del Instituto: Dra. Isar P. Godreau Directora Auxiliar: Sra. Vionex M. Marti

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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 g/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 antibacterial (1996, 2006)
6,7

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 g/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 g/mL using the brine shrimp assay. 9 The extracts with a LC50 1000

g/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 9-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 H3C

H OH CHO OH OCH3 O

O H3C

H OH CHO

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 -terpineol (6) among other of its respective derivatives (Figure 2). In 2001, Garca 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 -guaiaconic acid (9). (Figure 3).17

O O H3CO HO COOH COOH HO HO H 3C HO CH3 O OCH3 OH

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 H3CO NH H3CO HO NCH3

O crotonosine (10)

HO
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, antiinflammatory, 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 (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 (70230 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 Gunica, 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 Ro 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 L of solution measured with an automatic micropipette. The sample (0.002g) to be tested was dissolved in 100 L of DMSO and diluted with seawater (1900 L) 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 L of DMSO in 950 L of seawater. The bioassay was conducted in a 96 microwell plate. Several solutions of each sample ranging from 1.95 g/mL to 500 g/mL were prepared in triplicate. To each solution 100 L 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 -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 Aliphatic (0.2-1.5) Allylic X=C-CH3 (1.5-3.0)
-Heteroatoms

Vinylic C=C-H (4.5-5.5)

Aromatic Ar-H (6.5-8.0)

Species

H-C-Y (3-4)

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 COM/MPO 14

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 g/mL. The most promising activity was displayed by crude e xtracts of Simarouba tulae, Guaiacum officinale and Croton discolor with lethality values of 2, 21 and 111 g/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. COM/MPO 16

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 Canella winterana Extract Crude Hexane Chloroform Ethyl Acetate Crude Hexane Chloroform Ethyl Acetate Crude Hexane Chloroform Ethyl Acetate Crude Hexane Chloroform Ethyl Acetate Crude Hexane Chloroform Ethyl Acetate Crude Hexane Chloroform Ethyl Acetate Crude Hexane Dichloromethane Ethyl Acetate LC50 value in g/mL >200 78 >200 >200 111 132 >200 >200 >200 91 188 >200 21 87 3 4 >200 47 86 189 2 >200 161 35 >200 >200 >200 >200

Croton discolor

Gotzea elegans

Guaiacum officinale

Pimenta racemosa

Simarouba tulae

Thouinia striata

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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.

References
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Years J. Nat. Prod., 2007, 70, 461-477.

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the Caribbean Medicinal Resources of the Tropical Forest. Columbia University Press, New York. 1996.

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Melndez, P. A.; Capriles, V. A. Antibacterial Properties of Tropical Plants from Puerto Rico

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Antibacterial Activity Puerto Rico Health Sci. J., 1993, 12, 259-262.
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Chvez, P. I.; Snchez, L. A.; Gonzalez, F. A.; Rodriguez, J. L.; Axelrod, F. Cytotoxic

Correlations of Puerto Rican Plants Using a Simplified Brine Shrimp Lethality Screening Procedure Pharmaceutical Biology, 1997, 35, 222-226.
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Farouk S.; El-Feraly.; McPhail, A. T.; Onan, K. D. X-Ray crystal structure of canellal, a

novel antimicrobial sesquiterpene from Canella winterana J. Chem. Soc., Chem. Commun., 1978,75-76.
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Activity Relationships of Phytotoxic Sesquiterpenoids from Canella winterana J. Agric. Food Chem., 1995, 43, 826829.
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Fernndez, A.; Alvarez, A.; Garca, M. D.; Saenz, M. T. Anti-inflammatory effect of Pimenta

racemosa var. ozua and isolation of the triterpene lupeol IL Farmaco, 2001, 56, 335-338.
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Saenz, M. T.; Tornos, M. P.; Alvarez, A.; Fernndez, M. A.; Garca, M. D. Antibacterial

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

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Canella winterana
Classification
Kingdom Subkingdom Superdivision Division Class Subclass Order Family Genus While the outer bark is gray the inner bark is yellow and aromatic with the smell of mixed spices. Species Plantae
Tracheobionta
Spermatophyta

Canella winterana it is also known as canella alba, wild cinnamon and barbasco

Magnoliophyta Magnoliopsida Magnoliidae Magnoliales Canellaceae

General Information
Canella winterana, also known as wild cinnamon is a salt tolerant evergreen shrub. It has an open canopy that reaches a height of 10 meters with a diameter of 8 inches. Purple and white showy flowers cover the tree in summer and fall followed by bright red berries clustered near the tips of the branches.

Canella Winterana

The generic name is reputed to originate from the Latin for cinnamon as it is used in Spanish canela. The species epithet honors a captain Winter its who bark first to introduced Europe.

Distribution

This species occurs naturally from south word Florida through the West Indies and has been introduced in Brazil and Venezuela.

Traditional Uses
In folk medicine, the inner bark tea is used to treat fevers, relieve indigestions and is gargled to treat inflamed tonsils. Externally, the leaves are applied to relieve rheumatism and headache. The rum extract of the bark is used as a liniment to relieve rheumatism and other pains, and its liquor is consumed to treat stomach pains. On distillation the bark yields a volatile oil containing benzoyleugenol, mannitol, caryophyllene resins, canellal, cineol, clovanidiol, helicid, myristicin, 1-pinene, drimane sesquiterpenes, among other compounds. Canellal, isolated from bark is and non warburganal identified

antifungal, antimicrobial, cytotoxic, and inhibits insect feeding. The bark and leaves are still used in the West Indies to spice beverages and season food. The volatile oils found in the leaves have also been used as additives in perfumes.

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

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Croton discolor
Classification
Kingdom Subkingdom Superdivision Division Class Subclass Order Family Genus Species Variety methylcrotonosine, from the plant. linearisine and Plantae
Tracheobionta

This plant is also called white maran.

Spermatophyta
Magnoliophyta Magnoliopsida Rosidae Euphorbiales Euphorbiaceae

Croton L. Discolor willd (lechecillo)

General Information
Croton discolor is an aromatic 8 ft. tall 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, 8,14dihydrosalutadirine, The genus of over 600 species of is distributed worldwide and includes aromatic herbs, shrubs and trees. discolorine, which have been isolated

The species name is Latin and it refers to the fact that the top and the bottom of its leaves are of different color.

Distribution

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

found to promote tumors in mouse previously treated doses subcarcinogenic carcinogens. Croton discolor from Isla de Mona 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

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Goetzea elegans
Classification

Goetzea elegans is an endemic plant of Puerto Rico also known as beautiful, Matabuey and Manzanilla.

Kingdom Subkingdom Superdivision Division Class Subclass Order Family Genus Species

Plantae
Tracheobionta

Spermatophyta
Magnoliophyta Magnoliopsida

Asteridae Solanales Solanaceae

Goetzea Wydler.

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 semievergreen 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 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.

Goetzea elegans Wydler

Solanaceae family
In the solanaceae family the leaves can be opposite, clustered or alternate. There is also variety in the arrangement and the form of the flowers, but generally the corolla is radially symmetrical and five lobed with five stamens. The calyx and corolla can be either bell-shaped, wheel-shaped or cylinder-shaped.

Atropine has been isolated from several species of the Solanaceae family. It is a secondary metabolite that serves as a drug with a wide variety of effects. It is a competitive antagonist for the muscarinic acetylcholine receptor.

The Solanaceae family includes the potatoes, tomatoes, peppers, tobacco and petunias.

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
Tracheobionta

Guaiacum officinale is also known as the tree of life, it is the source of the true lignum vitae, a trade wood also called guayacan.

Subkingdom Superdivision Division Class Subclass Order Family Genus Species

Spermatophyta
Magnoliophyta Magnoliopsida

Rosidae Sapindales Zygophylaceae Guaiacum L. Guaicaum officinale L

General Information
Guaiacum officinale is an ornamental evergreen tree with pretty rich blue flowers, the trunk is a greenish-brown color, the wood of slow growth but attains a height of 40 to 60 feet, stem almost always crooked, bark furrowed; the wood is extraordinarily heavy, solid and dense, leaves, fibers oval cross-grained; obtuse; seeds fruit solitary, pinnate

heated, when it emits an agreeable scent. This wood was once very important for uses requiring strength, weight, and hardness. All species of the genus are now listed in CITES Trade (Convention in on
R1O

COOR2

International

Endangered

Species of Wild Fauna and Flora) as potentially endangered species. The bark yields 1 per cent volatile oil of delicious fragrance.
R1= azcares R2= H o azucares

Saponins form G. officinale

obcordate

capsule;

hard, oblong. The wood has a slight acrid taste and is odorless, unless

Distribution
This species occurs naturally in Florida Puerto Rico and the Virgin Islands. It is believed that the guaiacum did not had power to cure the syphilis but rather alleviate the symptoms. A number of saponins have been reported from different plant parts. Guaicins A to G have been found in leaves, stem bark and fruits. Heartwood contains lignans such as furoguaiacidin and furoguaiaodin.

Traditional Uses
Guaiacum resin is an acrid stimulant. It has been used to treat rheumatism, chronic skin diseases, scrofula and to prevent gout. As a remedy for venereal diseases guaiacum wood was successfully used. Nine years after its introduction to Europe more than three thousand persons had benefit from its therapy

Guaiacum officinale is the national flower of Jamaica.

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.

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Pimenta racemosa
Pimenta racemosa is also known as malagueta, guayabita, wild cinnamon and bay rum tree.
Classification
Kingdom Subkingdom Superdivision Division Class Subclass Order Family Plantae
Tracheobionta

Spermatophyta
Magnoliophyta Magnoliopsida

Rosidae Myrtales Myrtaceae Pimenta Lindl. Pimenta racemosa

General Information
Pimenta racemosa (Bay rum tree) is native to Puerto Rico and perhaps to the Virgin Islands and the other West Indian islands. The tree is identified by its opposite, smooth-margined, dark green leaves, which when crushed have a strong bay rum (clove-cinnamon) fragrance. Its bark is gray to light brown, white flowers borne in round clusters and the fruits are small, elongated green fruits that turn black in maturity. One of the varieties that have been described is the

Genus grisea with its oil rich in geraniol, methyl eugenol and isoeugenol. The citriodora variety has strongly lemon-scented oil consisting mostly of geranial. A believed chemovariety chavicol and produces methyl an aniseeugenol. scented oil consisting mostly of methyl Harvesting and extraction of any variety with true bay rum produces an oil of inferior quality. Species

Eugenol is the main constituent of bay leaf oil

Distribution
Pimenta racemosa can be found in Puerto Rico and the Virgin Islands.

Traditional Uses
The leaves have been used as a spice in cooking, as a perfume, and as a liniment to ease muscle aches, P. racemosa var. terebinthina is used in the Caribbean basin for different afflictions. In the Dominican Republic, the essential oil extracted from the leaves is used for the treatment used for of its rheumatism or for and toothache. P. racemosa var. grisea is anti-inflammatory analgesic properties.

Bay Leaf Oil

Bay leaf oil or myrcia oil is yellow to dark brown. The crude oil has a sweet Approximately 80 been detected have penetrating odor. compounds

including monoterpenes, sesquiterpenes, aromatic and aliphatic compounds. The major use for bay oil is in hair lotions with minor uses in perfumery and as a food flavoring.

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
Tracheobionta

Simarouba tulae is an endemic plant of Puerto Rico commonly known as aceitillo falso

Subkingdom Superdivision Division Class Subclass Order Family Genus Species

Spermatophyta
Magnoliophyta Magnoliopsida

Rosidae Sapindales Simaroubaceae

Simarouba Aubl.

Simarouba tulae Urb

Simaroubaceae Family
This family consists of approximately 25 genera and 150 species that are known by their chemical composition that includes oxygenated triterpenes such as the quassinoids. Usually their trees are slender, with long pinnate leaves on the top of a slender hole and racemes of little berries.

Traditional Uses
The bark, wood and leaves from the genus Simarouba have been used for their analgesic, antimalarial, sudorific, Its bark antimicrobial, astringent, antibacterial properties.

In the early twentieth century quassinoids were introduced in Europe as nicotine substituent.

antidysenteric, amoebicidic and has been used as a remedy for the treatment of diarrhea, dysentery and dyspeptic affections.

Quassinoids
The main active compounds of Simarouba are the quassinoids, which belong to the triterpene chemical family and which are responsible for its bioactivity and are considered chemical markers of the family.

O
There are about 100 quassinoids known and some like isobruceine A posses a strong antileukemic activity. Some other quassinoids have antiviral, antimalarial, antifeedant and insecticidal properties.

Basic skeleton of a Quassinoid C-20

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.

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Thouinia striata
Classification

Thouinia striata is an endemic plant found only in the island of Puerto Rico

Kingdom Subkingdom Division Class Subclass Order Family Genus Species Variety

Plantae
Tracheobionta Magnoliophyta Magnoliopsida Rosidae Sapindales Sapindaceae

Thouinia striata portoricensis striata

General Information
Thouinia striata is also known as dicotyledons. Thouinia striata pertains to the Sapindaceae botanical family, otherwise known as the Soapberry family. ceboruquillo and serrasuela. This tree and shrub is an endemic plant of Puerto Rico. Ceboruquillo is part of the Magnoliophyta division otherwise known as flowering plants. This plant is also a member of the magnoliopsida class usually referred to as

The common name of the genus refers to the presence of saponins in the soft pulps of the trees fruits. Solutions made from this pulp produce lather very

Sapindaceae family
Sapindaceae, soapberry flowering genera also is in known a the as the of order species, Many are lactiferous, i.e. they contain milky sap, and many contain mildly toxic saponins with soap-like qualities in the foliage and/or the seeds, or roots. Some of the sapindaceae species are of economic importance and including fish stimulating edible fruits, medicinal plants, poisons, beverages. soaps family, plants with family

much like the one produced by manufactured soap. Some species of the family have been used as detergents in tropical regions.

Sapindales. There are about 140-150 1400-2000 including maple, horse chestnut and lychee. Sapindaceae members occur in temperate to tropical regions throughout the world.

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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