Escuela de Farmacia, Recinto de Ciencias Médicas, Universidad de Puerto Rico

Bioactivities of latexes from selected tropical plants

Dr. Ricardo O. Guerrero1y Dr. Ángel L. Guzmán2

Summary

Fifteen latexes from selected tropical plants were collected in Puerto Rico, Guadalupe and Ecuador and evaluated by 2 bioassays. The tests carried out were the brine shrimp lethality test (BSLT) and the DNA-methyl green (DNA-MG) interaction. An additional assay, antibacterial activity, was performed on the bark/latex extract of Mammea americana L. The results indicated that some of these latexes are bioactive. On the BSLT assay, the latexes of Euphorbia neriifolia L. and Sapium laurocerasus Desf. displayed LC50 values of 76,7 and 7,1 µg/mL, respectively. Moreover, the dichloromethane (DCM) fraction of the bark/latex of M. americana L., presented a LC50 1,1 mg/mL. In addition, in the DNA-MG interaction, this particular fraction exhibited an IC50 211,8 mg/mL, whereas the latex of Croton menthodorus Benth. proved active with a IC50 390,2 mg/mL. Furthermore, the antibacterial assay on the methanol (MeOH) and DCM fractions of the bark/latex extract of M. americana L. indicated activity against several pathogenic bacteria. The positive results on these bioactive latexes should encourage the further investigation of the active principles responsible for these activities.

Subject headings: PLANTS, MEDICINAL; PLANT EXTRACTS; DNA; METHYLGREEN; PUERTO RICO; GUADALUPE; ECUADOR; BIOLOGICAL ASSAY.

Latex is considered to be the milky exudate of many plants that coagulate upon air exposure. The chemical composition of latex is very complex. It is composed of proteins, alkaloids, starches, sugars, oils, tannins, resins, gums, among other compounds. Most of the time latexes are white, although sometimes they are yellow, scarlet, or other colors. Several of the latexes have developed into substances of economic importance: rubber (Hevea sp.), chicle (Manilkara sp.), gutta-percha (Palachium sp.), etc. Among the latexes of biological importance, the medical usefulness of opium is amply recognized. The Euphorbiaceae, Moraceae, Cannabinaceae, Apocynaceae, and Asclepidiaceae families include many laticiferous species.1 Many of these plants are wild and tropical while a few are cultivated. Although some latex containing plants are nontoxic, most of them cause either irritant or contact dermatitis, and as a preventive measure, they all should be avoided.

There are more than 2 500 species of plants that produce natural latex (Wood M. Sunflower rubber? Agricult Res 2002; 22.). Most of these latexes remain to be scrutinized for their biological usefulness. As mentioned above, the objective of this study was to evaluate with 2 bioassays, 15 latexes of tropical plants from Puerto Rico, Guadeloupe, and Ecuador. The bioassays are:

1. The brine shrimp lethality bioassay in microplate (BSLT), a general toxicity evaluation of biological samples, and 2. The interaction with DNA-methyl green (DNA-MG), which detects agents or metabolites that link up with DNA. In addition to these 2 bioassays, an antibacterial examination was conducted with the bark/latex of Mamea americana L.

Methods

During the summer of 1995, 5 latexes were collected in Ecuador by Dr. Victor Hugo Villacrés and Mr. Valdano Tafur, former members of the "Instituto de Ciencias Naturales" of the "Universidad Central del Ecuador". In the island of Guadaloupe and in the same year, 1 latex sample was collected by pharmacist Joseph Henry. In Puerto Rico, 8 latexes were collected by Dr. Julio Figueroa of the Forest Service, Department of Agriculture (See Table I). The plants from which the latexes were obtained have been preserved for future reference in the Herbarium of the "Instituto de Ciencias Naturales" of Ecuador, and the Herbarium of the Botanical Garden of San Juan, Puerto Rico. The latexes were collected in brown containers, maintained in the dark and refrigerated as soon as it was possible. One of the samples, the latex of Mammea americana L. was obtained together with the bark. In this particular case, the plant material was extracted with ethanol (95 %) and partitioned between MeOH and DCM.

Brine shrimp lethality test in microplate (BSLT)

This is a general bioassay that detects a variety of toxic substances and has been applied to plant extracts since 1982.2 Usually, the results of this test correlate with cytotoxicity and pesticidal activity. Latex samples (1 mg), were dissolved in 50 µL of DMSO and 950 mL of sea water was added. Dissolution was assisted with a sonicator and if the sample failed to dissolve completely, it was filtered through Millex-AA Particulate Filter Unit (Millipore, Bedford, MA, USA).

The little crustacean Artemia salina Leach was used in this bioassay. The protocol described by Solís et al.3 1993, was followed in our study. Brine shrimp eggs were acquired in a pet shop (San Juan, Puerto Rico) and hatched in sea water (Instant Ocean, Aquarium Systems, Mentor, Ohio, USA) at room temperature. After 48-72 hours the debris-free nauplii were collected. A suspension with 10 to 15 shrimp in 100 mL was added to each well containing 100 mL of the test solution at different concentrations and the covered microplate was placed under white light for 24 h at room temperature. After this period of incubation, the dead nauplii were counted (Bausch and Lomb binocular microscope, 20 X). 50 mL of MeOH was added to each well and after 30 minutes the total number of nauplii was recorded. The LC50 values and 95 % confidence intervals were determined in mg/mL, using the Finney probit analysis computer program. Artificial seawater was used as a negative control and Berberine chloride (Sigma, St. Louis, MO, USA) was employed as positive control. Extracts with LC50 £ 100 mg/mL were considered active.

DNA-Methyl Green Interaction, (DNA-MG).

This bioassay has been designed on the basis that certain clinically useful agents interact either covalently or non-covalently with DNA. These interactions may or may not reflect a cytotoxic response. This system uses the triphenyl methane methyl green dye. This dye binds the DNA and the DNA/methyl green reversible complex is commonly used as substrate to measure DNAase activity. When methyl green is displaced from DNA, a water molecule is added to methyl green resulting in the formation of a colorless molecule, carbinol. This reaction can be followed spectrophotometrically as a decrease in absorbance.4

In this bioassay, latexes samples (2,0 mg) were dissolved in 1 mL of ethanol and 20 mL of the sample solutions were placed into wells of a 96-well microtiter and the solvent was removed in vacuum. 20 mg of the reagent DNA-MG (Sigma, St. Louis, MO, USA) were suspended in 100 mL of 0.05 M Tris-HCl buffer, pH 7,5, containing 7,5 mM of magnesium sulfate and stirred at 37 °C during 24 hours. 200 mL of the above solution were added to each well. The initial absorbance of each sample was measured at 630 nm using a UV-Max Kinetic Microplate Reader from Molecular Device Corporation. The samples were incubated in the dark for 24 hours at room temperature. After this time, the final absorbance of the samples was measured as above. The readings were corrected for the initial absorbance and normalized as percentage of the untreated DNA/methyl green absorbance value. The IC50 and 95 % confidence values were determined as above for latexes that presented a substantial decrease in absorbance. Doxorubicine HCL (Sigma, St. Louis, MO, USA) was used as a positive control.

Antibacterial activity. As mentioned above, the bark/latex of M. americana were extracted with ethanol (95 %) and partitioned between DCM and MeOH. These two fractions were evaluated for antibacterial activity using the disk susceptibility testing.5 A battery of five human pathogenic bacteria served as test microorganisms: gram-positive Staphylococcus aureus (ATCC 25923) and Streptococcus pyogenes (ATCC 19615); and gram-negative Xanthomonas maltophilia (Medical Center Hospital sample), Citrobacter freundii (Medical Center Hospital sample), and Klebsiella pneumoniae (ATCC 13883). Some of the microorganism samples were purchased from Sigma, St. Louis, MO, USA. Tetracycline, clarithromycin and amoxicillin (Sigma, St. Louis, MO, USA) were used as positive controls. Both MeOH and DCM fractions of M. americana turned to be active toward these microorganisms. On this account, minimal inhibitory concentration (MIC) values were investigated with the micro titer plate method.6

Results and Discussion

In order to study the bioactivity of 15 latexes from tropical plants, 2 general bioassays were used: BSLT and DNA-MG. A third bioassay, the antibacterial activity, was conducted on the MeOH and DCM partitions of the ethanolic extract of the bark/latex of M. americana L. The BSLT and DNA-MG results are listed in Table I. In the BSLT bioassay, 3 latexes of the 15 collected (20 %), showed significant concentration related activity with LC50 values below 100 mg/mL: Euphorbia neriifolia L., LC50 77,00 mg/mL (154,0-43,0), Sapium laurocerasus Desf., LC50 7,00 mg/mL (9,2-5,0), and the DCM solubles of M. americana L., LC50 1.10 mg/mL (1.13-0.48). Berberine chloride, the positive control, exhibited LC50 166 M. According to these results, there is a good probability that metabolites of these plants may have cytotoxic and/or pesticidal activities.

TABLE 1. Bioassays screening of latexes from tropical plants

Entry	Scientific Name	Family	Common name	Origin	BSLT LC50 µg/mL	DNA-MeG Interaction IC50 µg/mL
1	Artocarpus altilis (S. Park) Fosb.	Moraceae	Panapén, Bread fruit	Puerto Rico	>1,000
2	Croton lechleri Muell. Arg.	Euphorbiaceae	Sangre de Drago, Dragon's blood	Ecuador	>1,000
3	Croton menthodorus Benth.	Euphorbiaceae	Mosquera	Ecuador	>1,000	390,2 (547,7-314,7)
4	Croton poecylanthus Urban	Euphorbiaceae	Sabinón	Puerto Rico	>1,000
5	Euphorbia cotinifolia L.	Euphorbiaceae	Barrabás, poison spurge	Ecuador	>1,000
6	Euphorbia hirta L.	Euphorbiaceae	Ecuador		>1,000
7	Euphorbia lactea Haw.	Euphorbiaceae Cauto, mottled spurge	Puerto Rico		>1,000
8	Euphorbia neriifolia L.	Euphorbiaceae Antena, hedge euphorbia	Puerto Rico			76,7 (154,2-43,3)*
9	Euphorbia tirucalli L.	Euphorbiaceae Esqueleto, pencil bush	Puerto Rico			180,0 (283,9-128,4)
10	Ficus benjamina L.	Moraceae Laurel, weeping fig	Puerto Rico		>1,000
11	Hippomane mancinella L.	Euphorbiaceae Manzanillo, manchineel	Puerto Rico			255,4 (523,1-157,6)
12	Mammea americana L. **	Guttiferae Mamey, mammee-apple	Puerto Rico			1,1 (1,3-0,9) 211,8 (338,1-92,3)
13	Pterocarpus officinalis Jacq.	Leguminosae Palo de pollo, swamp bloodwood	Guadaloupe		>1,000
14	Sapium laurocerasus Desf.	Euphorbiaceae Tabaiba	Puerto Rico			7,1 (9,2-5,0)
15	Sapium utile Preuss.	Euphorbiaceae Barbasco	Ecuador		>1,000

          *95 % confidence limits
          **Bark/latex, dichloromethane extract

In reference to previous investigations of the active plants, it was found that a lectin from the latex of E. neriifolia has been isolated and partially characterized. The lectin possesses misogynic activity with marine spleen lymphocytes, but it does not inhibit protein synthesis in rabbit reticulocyte lysate.6 Whether the BSLT activity is due to the lectin, it is not known. S. laurocerasus is an endemic plant of Puerto Rico.( Martorell LF, Liogier AL, Woodbury RO. Catálogo de los Nombres Vulgares y Científicos de las Plantas de Puerto Rico. Estación Experimental Agrícola, Río Piedras, Puerto Rico, 1981:81.) To date, no reports on its bioactivity are present in the scientific literature. As for M. americana L., the seed's oil has been investigarted.7,8 In these studies, an antitumor constituent, mammein, and some coumarin and phloroglucinol derivatives were isolated. In another study, several xanthones and benzophenones with antitumor and antibacterial activity were also isolated from the seed's oil.9 There is the possibility that these compounds might also be present in the bark/latex extract. In another investigation, it was reported that the leaves extract of this plant presented anti-Mycobacterium tuberculosis activity.10

Furthermore, it has been published that M. americana leaves extract was the most effective of 173 plant extracts for its molluscicidal properties against Bioamphalaria glabrata.11 Finally, a very recent investigation has shown that the DCM fraction of the bark/latex extract presented anti-ulcerogenic activity in rats.12

A colorimetric bioassay for the detection of agents that interact with DNA was also carried out with the DNA-MG reagent. The results (Table 1) indicate that 2 latexes of the 15 collected (13,33 %) were bioactive, indicating the presence of compounds that interact with DNA. IC50 values for Croton menthodorus and Mammea americana (DCM fraction) were 390,2 mg/mL and 211,8 mg/mL, respectively. The positive control, doxorubicine HCl had 75,04 % absorbance decrease at 4,31 x 10-4 N.

In relation to previous studies on C. menthodorus, it has been reported that several quercetin, rutin and kaempferol glycosides isolated from the plant extract reduce morphine withdrawal in vitro.13 In another investigation, a compound identified as morphinandien-7-one, O-methyl flavinantine, was identified from polar extracts of this plant. This metabolite significantly reduced the electrical contractions of the guinea-pig isolated ileum.14 No studies on the latex of this plant appear in the literature.

The results of the antibacterial activity on the DCM and MeOH fractions of M. americana are shown in Table 2. The MeOH fraction was fairly active against gram-positive microorganisms: S. aureus and S. pyogenes, (MIC 6,25 mg/mL for both bacteria). The MeOH fraction was not as effective against the gram-negative bacteria: K. pneumoniae, C. freundii and X. maltophilia. The DCM fraction presented excellent activity for gram-positive and gram-negative bacteria with MIC values ranging from 12,5 mg/mL for S. pyogenes to 25 mg/mL for the other microbes. These values are very encouraging to support subsequent studies on the identification of the antibacterial compounds.

TABLE 2. Antimicrobial activity of bark/latex from mammea americana L. Mic values (µg/mL)

Mammea americana L.	Staphylococcus aureus	Streptococcus pyogenes	Citrobacter freundii	Klebsiella pneumoniae	Xanthomonas maltophylla
DCM fraction	25	12,5	25	25	25
MEOH fraction	6,25	6,25	50	50	50

This work has confirmed biological activities of several latexes from selected tropical plants. It is expected that these positive results will serve as a motivation for further examination of the active principles.

Acknowledgements

The authors wish to express their appreciation to Dr. Victor H. Villacrés, Mr. Valdano Tafur, pharmacist Joseph Henry and Dr. Julio Figueroa for the identification of the plants and collection of the latexes; to Mrs. Slavomira A. Kucerova for conducting the antibacterial study; to Dr. Mikhail Antoun and Mrs. Zulma Ramos for technical assistance, and to Mrs. Miriam Corazón, Director Bacteriology Department (Clinical Laboratory), Medical Center Hospital, for the identification and submission of some of the microorganisms samples. ALG appreciates the support of the San Juan Bautista School of Medicine.

Resumen

Quince látexes provenientes de plantas tropicales selectas fueron coleccionados en Puerto Rico, Guadalupe y Ecuador, y evaluados con 2 bioensayos. Los ensayos llevados a cabo fueron el examen de mortalidad de los camarones salinos y la interacción entre DNA y verde de metilo. Un ensayo adicional, la actividad antibacteriana, fue realizada con el extracto de la corteza/látex de Mammea americana L. Los resultados indicaron que algunos de estos látexes son bioactivos. En el ensayo de mortalidad de los camarones salinos, los látexes de Euphorbia neriifolia L. y Sapium laurocerasus Desf., mostraron valores de LC50 de 76,7 y 7,1 mg/mL, respectivamente. Además, la fracción de diclorometano de la corteza/látex de M. americana L., presentó LC50 de 1,1 mg/mL. En adición, en la interacción entre DNA y verde de metilo, esta fracción particular exhibió IC50 de 211,8 mg/mL, mientras que el látex de Croton menthodorus Benth, probó ser activo con IC50 de 390,2 mg/mL. Además, el ensayo antibacteriano de las fracciones de metanol y diclorometano del extracto de la corteza/látex de M. americana L., indicó actividad contra algunas bacterias patógenas. Los resultados positivos de estos látexes bioactivos deben servir de aliciente para investigar los principios activos responsables de estas actividades.

Palabras clave: Plantas medicinales; extractos vegetales; adn; verde de metilo; puerto rico; guadalupe; ecuador; bioensayos.

References

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Recibido: 24 de noviembre de 2003. Aprobado: 5 de diciembre de 2003.
Dr. Ricardo O. Guerrero. University of Puerto Rico. P.O. Box 5067, San Juan, PR 00936-5067. Tel [phone] x 5418; Fax [phone].E-mail: [e-mail]

1Ph.D. in Pharmacology.
2 Ph.D. Biochemistry.