Abstract
We used an alkaloid test and a brine shrimp bioassay to assess the bioactivity of the medicinal plants used by eastern Nicaraguan healers in traditional medicine. Ethnomedicinal uses were obtained from interviews of traditional healers. Aqueous extracts derived from 30 species of angiosperms were assayed for the presence of alkaloids and toxicity. Species tested are distributed in 30 genera and 21 families. Of the 30 species tested for alkaloids with Dragendorff’s reagent, 29 contained alkaloids. Toxicological analysis was conducted using the brine shrimp lethal assay (BSLA). Biological activity using BSLA was recorded as the median lethal concentration (LC50) that kills 50% of the larvae within 24 h of contact with the aqueous plant extracts. The LC50 of the shrimp was less than 2500 µg/mL for 3 (10%) species, 2500-5000 µg/mL for 9 (30%), 5001-7500 µg/mL for 7 (23%), 7501-10000 µg/mL for 3 (10%), and greater than 10000 µg/mL for 8 (27%) of the species. The members of the orders Santales and Rubiales in general contained species with greater toxicity than any other group. Struthanthus cassythoides (Struthanthus cassythoides Millsp.(Loranthaceae)). (LC50 1574 µg/mL) and Alibertia edulis (Rich.) A. Rich. (Rubiaceae) (LC50 1741 µg/mL) were the most toxic.
Introduction
Medicinal plants contribute greatly to primary healthcare in many parts of the world. In rural areas of Central America, ethnomedicines are the primary source of healthcare. Recently, traditional medicine has gained greater acceptance by the urban population as an alternative form of healthcare, and phytomedicine has become an important source of income for many people. Herbal remedies prepared with ethnomedicinal species are used to treat a vast array of ailments in eastern Nicaragua, ranging from fever to snakebites (CitationDennis, 1988; CitationBarrett, 1994; CitationCoe, 1994; CitationCoe & Anderson, 1996a, Citation1996b, Citation1997, Citation1999, Citation2005) but there is scarce scientific evidence confirming their efficacy or toxicity. For these reasons, medicinal plants have become the focus of intense study to validate traditional uses via determination of pharmacological effects (CitationStone, 2008).
The toxicity of crude extracts of medicinal plant species from several countries has been assessed, with examples from Argentina (CitationBustos et al., 1996; CitationHernández et al., 2000), Bahrain (CitationTaha & Alsayed, 2000), Bangladesh (CitationCosta-Lotufo et al., 2005), Brazil (CitationHammer & Johns, 1993; CitationAlves et al., 2000; CitationHoletz et al., 2002; CitationSantos Pimenta et al., 2003; CitationQuignard et al., 2004; CitationFerreira de Lima et al., 2006; CitationSuffredini et al., 2006; Citationdos Santos et al., 2007), Canada (CitationMcCuthcheon et al., 1992), Chile (CitationCuadra et al., 2005), China (CitationDuffy & Power, 2001), Cuba (CitationMartinez et al., 1996; CitationLogarto Parra et al., 2001), Ecuador (CitationGuerrero et al., 2003), Guatemala (CitationCáceres et al., 1993a, Citation1993b, Citation1998; CitationMichel et al., 2007), Honduras (CitationLentz et al., 1998), India (CitationPadmaja et al., 2002), Kenya (CitationWanyoike et al., 2004), Mexico (CitationSánchez-Medina et al., 2001), Nigeria (CitationAjaiyeoba et al., 2006), Panama (CitationSánchez et al., 1993), Papua New Guinea (CitationNick et al., 1995), the Philippines (CitationHorgen et al., 2001), Uruguay (CitationGonzález et al., 1993), and the USA (CitationTurker & Camper, 2002; CitationBadisa et al., 2007; CitationVan Slambrouck et al., 2007). However, phytochemical screening and bioassays of crude extracts of medicinal plants are lacking in many regions of the world where dependency on herbal remedies is high.
The flora of the Eastern Lowlands of Nicaragua consists of over 2,890 species, a number that is about half of the total flora (estimated at about 5,800 species) of vascular plants (CitationSutton, 1989; CitationStevens et al., 2001). Over 350 of the species of vascular plants of the Eastern Lowlands are used as medicines (CitationCoe, 1994; CitationCoe & Anderson 1996a, Citation1996b, Citation1997, Citation1999, Citation2005). However, very little is known about the chemistry and toxicity of these medicinal plants. Screening of medicinal species for bioactive compounds in Eastern Nicaragua is limited to studies by CitationCoe (1994) and CitationCoe and Anderson (1996a, Citation1996b, Citation1997, Citation1999, Citation2005). Similarly, bioassay studies to determine the toxicity of these medicinal species are also lacking. We chose ethnomedicinal species for these bioassays because it is widely recognized that the success rate of finding bioactive compounds from these species is much greater than from random collecting or focusing on particular plant families (CitationFarnsworth & Soejarto, 1985; CitationFarnsworth, 1988; CitationTyler et al., 1988; CitationCox, 1994; CitationCoe & Anderson, 1996b). We focused on alkaloid-bearing species because alkaloids are known to be highly bioactive, and this would suggest that they could be toxic to brine shrimp. We report the results of alkaloid tests and brine shrimp bioassays of plant extracts of 30 species currently used in traditional medicine by healers in eastern Nicaragua.
Materials and methods
Plant collection
Plant material was collected during field trips with traditional healers (shamans, midwives, and herbalists) during several months over many years (1992-2007). Further details about these ethnobotanical studies are published elsewhere (CitationCoe, 2008a,Citationb). Voucher specimens were deposited at the Missouri Botanical Garden (St. Louis, MO) and the University of Connecticut (Storrs, CT). Vouchers were identified by F. Coe with the assistance of specialists listed in the Acknowledgements.
Thirty medicinal species were screened for alkaloids (). Plant parts tested are identical to those used in the preparation of herbal remedies, and included both aerial and underground parts (). Plant materials were obtained from mature individuals in flower or fruit, and collected and processed according to standard practices (CitationLawrence, 1951; CitationBridson & Forman, 1992; CitationSoejarto, 1993; CitationSoejarto et al., 1996). Alkaloid tests were performed in the same way in the field and in the laboratory, using Dragendorff’s reagent (CitationHarborne, 1988) and thin-layer chromatography (TLC) (CitationStahl, 1969). Alkaloids were determined qualitatively by macerating 10-15 mg of plant material in a test tube in 1-2 mL of 1M Na2CO3. Once macerated, 0.5-1 mL of 2:1 CHCl3-MeOH was added. The mixture was mixed with a stirring rod for 3-5 min, and then allowed to stand and separate into two phases (upper and lower). The lower phase contained CHCl3 , and the plant extract was drawn off with a disposable pipette into a depression in a spot plate. The CHCl3 was allowed to evaporate to about a drop (0.025 mL). This concentrate was then spotted on an aluminum backed thin layer chromatography strip 10 mm × 40 mm in size. The strips were developed in CHCl3 and alkaloids were visualized (color ranges are yellow/orange, red/orange, red/black, pink, and even purple depending on the species or genus) by spraying with Dragendorff’s reagent. Alkaloids were considered present in a plant part when at least two of three replicates gave positive results. We are aware that field alkaloid tests can sometimes produce false-positive reactions, especially in latex bearing families, e.g., Apocynaceae, Araceae, Clusiaceae, Convolvulaceae, and Moraceae (CitationFarnsworth, 1966). The method we used (CitationStermitz et al., 1989) includes a purification procedure (adding a base-Na2CO3 and extraction with a water immiscible organic solvent-CHCl3) that helps avoid false-positive reactions. However, we verified our test results, where possible, with reports in the literature.
Table 1. Bioactivity and bioassay results of medicinal plants used in eastern Nicaragua.
Plant crude extract preparation
Plant crude extracts (stock solution) were prepared by boiling 1 g of plant material in 100 mL of distilled water as described by CitationBertani et al. (2007). An appropriate amount of the stock solution was mixed with 1% NaCl solution in vials to make concentrations of 500, 1,000, 2,500, 5,000, 7,500, and 10,000 µg/mL. Control brine shrimp larvae were placed in a mixture of 1% NaCl solution. Three replicates were prepared for each dose level. These relatively high doses were used to replicate the concentrations at which herbal remedies are prepared and administered by healers in eastern Nicaragua.
Hatching of brine shrimp
Eggs of brine shrimp (Artemia salina, Artemiidae) were purchased from Carolina Biological Supply (Burlington, NC) and were incubated for 48 h in a culture vessel (15 × 15 × 15 cm) containing saltwater (1% NaCl) prepared from nitrate, phosphate, and silicate-free sea salt and deionized water (35 g/L) at 24° to 28°C under a lamp. The saltwater solution was aerated continuously during incubation with an aquarium air pump (AirTech-2KO). After 48 h the nauplii (larvae) were collected from the culture vessel.
Brine shrimp lethality assay
The brine shrimp lethality assay (BSLA) was used to determine if the plant extracts of medicinal species were cytotoxic (CitationMeyer et al., 1982; CitationMcLaughlin et al., 1991; CitationCepleanu et al., 1994). Ten brine shrimp larvae were placed in each of the triplicate vials (thus, 30 shrimp per concentration) using a plastic pipette with a 2 mm diameter tip. The larvae were released under the surface of the solution to avoid killing them by trapping air under their carapaces. Survivors were counted under the stereomicroscope after 24 h, and the percentage of deaths at each dose and control was determined.
Data analysis
The mean results of brine shrimp mortality were plotted against the logarithms of concentrations using the computer program Probit Analysis Version 1.5 developed by the US Environmental Protection Agency from which median lethal concentrations (LC50) at 95% confidence intervals (CI) were calculated, according to the method of CitationFinney (1971). Biological activity using the brine shrimp bioassay was recorded as a lethal concentration when 50% of the larvae were killed within 24 h of contact with the extract. LC50 values greater than 1,000 µg/mL for plant extracts were considered inactive.
Results
The 30 species assayed belong to a diverse group of plants distributed in 30 genera and 21 families. The ethnomedicinal uses, bioactivity, and bioassay information of the 30 species assayed in this study are presented in and . Among the 30 species screened for alkaloids, 29 were positive (). A total of 30 plant extracts from 30 species were assayed for cytotoxicity to brine shrimp. All 30 species tested were toxic to brine shrimp above 1,000 µg/mL (). The concentration lethal to 50% of the shrimp was less than 2,500 µg/mL for 3 (10%) species, 2,500-5,000 µg/mL for 9 (30%), 5,001-7,500 µg/mL for 7 (23%), 7,501-10,000 µg/mL for 3 (10%), and greater than 10,000 µg/mL for 8 (27%) of the species (). The species with the highest cytotoxicity were Struthanthus cassythoides (Struthanthus cassythoides Millsp, Loranthaceae) (LC50 1,574 µg/mL) and Alibertia edulis (Rich.) A. Rich. (Rubiaceae) (LC50 1,741 µg/mL) (). The above results showed that these extracts were toxic to brine shrimp at LC50 values greater than 1,000 µg/mL, which can be considered inactive.
Table 2. Number of the 30 species assayed used for each medicinal application.
Discussion
The 30 species tested in this study are widely used by healers in eastern Nicaragua to treat over 23 ailments ( and ). Remedies prepared with these medicinal plants serve as treatment for a variety of ailments from common colds to snakebites. Nevertheless, very little is known about their toxicity. Recent tests of some popular herbal remedies have proven them very toxic, as is the case for Aristolochia fangchi (Aristolochia fangchi Y. C. Wu ex L. D. Chow & S. M. Hwang, Aristolochiaceae) (CitationGreensfelder, 2000; CitationNortier et al., 2000).
In this study, one species (Eugenia acapulcensis (Eugenia acapulcensis Steud., Myrtaceae)) tested negative for alkaloids, however, it is important to note that E. acapulcensis was cytotoxic (). The bioactivity of this species in the brine shrimp assay suggests that other types of secondary metabolites such as monoterpenes and sesquiterpenes (CitationVila et al., 2004), could be responsible for its cyto-toxicity. For example, the oil of this species exhibited strong antibacterial activity against Staphylococcus aureus and Mycobacterium smegmatis at 1,000 µg/mL (CitationVila et al., 2004). Therefore, the bioactivity of whatever the active compounds are is demonstrated, despite the absence of a positive alkaloid test.
The highest cytotoxicity recorded was in a species that tested positive for alkaloids, causing brine shrimp death (LC50) at concentrations below 2,000 µg/mL (). In contrast, some species that tested positive for alkaloids were cytotoxic (causing LC50) at concentrations greater than 10,000 µg/mL (). The degree of cytotoxicity was found to be directly proportional to the concentration of the extract. Maximum cytotoxicity occurred at concentrations equal to or greater than 5,000 µg/mL (), whereas minimum cytotoxicity was present at concentrations less than 2,500 µg/mL (). Although LC50 occurred at concentrations greater than 1,000 µg/mL for all extracts using the brine shrimp assay, this does not preclude the effective uses of these plants as effective medicines. The presence of alkaloids and of other bioactive compounds, such as tannins and flavonoids (CitationDiaz et al., 1988), glycosides (CitationChukwurah & Ajali, 2000) and saponins (CitationPretorius et al., 2003) might help to explain the use of these medicinal species by healers. For example, in this study the aqueous extracts of Hyptis verticillata (Hyptis verticillata Jacq. (Lamiaceae)) were considered inactive because they had a LC50 value of 3,775 µg/mL, but in a study by CitationLentz et al. (1998) the ethanol extracts of this species were toxic to the bacterium Pseudomonas aeruginosa and the fungus Trichophyton mentagrophytes. Similarly, Manihot esculenta aqueous extract in our study was inactive with a LC50 value of 5,956 µg/mL; however, results obtained by CitationZakaria et al. (2006) using chloroform and ethanol extracts showed strong antibacterial activity to Gram positive (Corneybacterium diphtheriae, Listeria monocytogenes, Pseudomonas aeruginosa) and Gram negative (Vibrio cholerae, Shigella flexneri and Salmonella typhi) bacteria.
The members of the orders Santales and Rubiales in general contained species with greater toxicity than any other group. The species with the highest cytotoxicity were members of the family Loranthaceae and Rubiaceae. However, the Loranthaceae is not known to contain species rich in bioactive compounds; conversely the Rubiaceae is highly regarded for its numerous species that are rich in bioactive compounds.
Given that these extracts tested positive for alkaloids and because the bioassay results showed toxicity to brine shrimp only at very high concentrations, perhaps additional testing, using other organisms and different types of extraction solvents, is needed to further assess the efficacy of these medicinal species.
Acknowledgements
Thanks to the Garífuna, Miskitu, Rama, and Sumu people for sharing their ethnobotanical knowledge. The field assistance of Basilio Benjamin, Far Blanford, Dale Desousa, Rodney Martin, and Harry Simmons, Jr. is appreciated. The assistance of the staffs of CIDCA (Centro de Investigación y Documentación de la Costa Atlantica) and FADCANIC (Fundación Para la Autonomía y Desarrollo de la Costa Atlántica de Nicaragua) is also appreciated. Many specialists provided assistance in the identification of vouchers: Daniel Austin (Arizona-Sonora Desert Museum), Gerrit Davidse (MO), Ronald Leisner (MO), Amy Pool (MO), Warren D. Stevens (MO), and Charlotte M. Taylor (MO). Thanks to Irsa Amin and Kellyn Misset for help in the laboratory, and Allison Robinson for numerous comments on drafts of the manuscript.
Declaration of interest
This study was partially supported by grants from the National Science Foundation, The University of Connecticut Research Foundation and the Department of Ecology and Evolutionary Biology.
References
- Ajaiyeoba EO, Abiodun OO, Falade MO, Ogbole NO, Ashidi JS, Happi CT, Akinboye DO (2006): In vitro cytotoxicity studies of 20 plants used in Nigerian antimalarial ethnomedicine. Phytomedicine 13: 295–298.
- Alves TM, Silva AF, Brandão M, Grandi TS, Smânia E, Smânia Júnior A, Zani CL (2000): Biological screening of Brazilian medicinal plants. Mem I Oswaldo Cruz 95: 367–373.
- Badisa RB, Badisa VL, Walker EH, Latinwo LM (2007): Potent cytotoxic activity of Saururus cernuus extract on human colon and breast carcinoma cultures under normoxic conditions. Anticancer Res 27: 189–193.
- Barrett B (1994): Medicinal plants of Nicaragua’s Atlantic coast. Econ Bot 48: 8–20.
- Bertani S, Houël E, Bourdy G, Stien D, Jullian V, Landau I, Deharo E (2007): Quassia amara L. (Simaroubaceae) leaf tea: Effect of the growing stage and desiccation status on the antimalarial activity of a traditional preparation. J Ethnopharmacol 111: 40–42.
- Bridson GDR, Forman L (1992): The Herbarium Handbook. London, Royal Botanic Gardens, Kew, 303 pp.
- Bustos DA, Tapia AA, Feresin GE, Ariza Espinar L (1996): Ethno-pharmacobotanical survey of Bauchazeta district, San Juan Province, Argentina. Fitoterapia 67: 411–415.
- Cáceres A, Figueroa L, Taracena AM, Samyoa B (1993a): Plants used in Guatemala for the treatment of respiratory diseases. 2: Evaluation of activity of 16 plants against Gram-positive bacteria. J Ethnopharmacol 39: 77–82.
- Cáceres A, López B, Juarez X, del Aguila J, García S (1993b): Plants used in Guatemala for the treatment of dermatophytic infections. 2. Evaluation of antifungal activity of seven American plants. J Ethnopharmacol 40: 207–213.
- Cáceres A, López B, González S, Berger I, Tada I, Maki J (1998): Plants used in Guatemala for the treatment of protozoal infections. I. Screening of activity to bacteria, fungi and American trypanosomes of 13 native plants. J Ethnopharmacol 62: 195–202.
- Cepleanu F, Hamburger MO, Sordat B, Msonthi JD, Gupta MP, Saadou M, Hostettmann K (1994): Screening of tropical medicinal plants for molluscicidal, larvicidal, fungicidal and cytotoxic activities and brine shrimp toxicity. Int J Pharmacog 32: 294–307.
- Chukwurah BKC, Ajali U (2000): Antimicrobial investigation of the constituents of the methanol extract of the rhizomes of Anchomanes difformis, Eng. Indian J Pharm Sci 62: 296–299.
- CIDCA (Centro de Investigación y Documentación de la Costa Atlántica) (1986): Diccionario elemental: Miskito-Español/Español-Miskito. Managua, Nicaragua, Centro de Investigación y Documentación de la Costa Atlántica, Ministry of Agriculture and Agrarian reform, 72 pp.
- CIDCA (1987): Diccionario Elemental Rama. Eugene, OR, University of Oregon, 109 pp.
- CIDCA (1989): Diccionario elemental del Ulwa: Sumu Meridional. Cambridge, MA, Centro de Ciencia Cognitiva, Massachusetts Institute of Technology, 165 pp.
- Coe FG (1994): Ethnobotany of the Garífuna of Eastern Nicaragua. Storrs, CT, Doctoral Dissertation (Botany), University of Connecticut, 292 pp.
- Coe FG (2008a): Ethnobotany of the Rama of southeastern Nicaragua and comparisons with Miskitu plant lore. Econ Bot 62: 40–59.
- Coe FG (2008b): Ethnomedicine of the Rama of southeastern Nicaragua. J Ethnobiol 28: 1–38.
- Coe FG, Anderson GJ (1996a): Ethnobotany of the Garífuna of eastern Nicaragua. Econ Bot 50: 71–107.
- Coe FG, Anderson GJ (1996b): Screening of medicinal plants used by the Garífuna of eastern Nicaragua for bioactive compounds. J Ethnopharmacol 53: 29–50.
- Coe FG, Anderson GJ (1997): Ethnobotany of the Miskitu of eastern Nicaragua. J Ethnobiol 17: 171–214.
- Coe FG, Anderson GJ (1999): Ethnobotany of the Sumu (Ulwa) of southeastern Nicaragua and comparisons with Miskitu plant lore. Econ Bot 53: 363–386.
- Coe FG, Anderson GJ (2005): Snakebite ethnopharmacopoeia of eastern Nicaragua. J Ethnopharmacol 96: 303–323.
- Costa-Lotufo LV, Khan MT, Ather A, Wilke DV, Jimenez PC, Pessoa C, de Moraes ME, de Moraes MO (2005): Studies of the anticancer potential of plants used in Bangladeshi folk medicine. J Ethnopharmacol 99: 21–30.
- Cox PA (1994): The ethnobotanical approach to drug discovery: strengths and limitations, in: Chadwick DJ, & Marsh J, eds, Bioactive Compounds from Plants, Ciba Foundation Symposium, no. 154. Chichester and New York, John Wiley, pp. 25-41.
- Cuadra P, Furrianca M, Oyarzún A, Yáñez E, Gallardo A, Fajardo V (2005): Biological activity of some Patagonian plants. Fitoterapia 76: 718–721.
- Dennis PA (1988): Herbal medicine among the Miskito of eastern Nicaragua. Econ Bot 42: 16–28.
- Diaz R, Quevedo-Sarmiento R, Ramos-Cormenzana, A (1988): Phytochemical and antibacterial screening of some spices of Spanish Lamiaceae. Fitoterapia 4: 329–333.
- dos Santos AF, de Azevedo DP, dos Santos Mata Rda C, de Mendonca DI, Sant’Ana AE (2007): The lethality of Euphorbia conspicua to adults of Biomphalaria glabrata, cercaria of Schistosoma mansoni and larvae of Artemia salina. Bioresource Technol 98: 135–139.
- Duffy CF, Power RF (2001): Antioxidant and antimicrobial properties of some Chinese plant extracts. J Antimicrob Agents 17: 527–529.
- Farnsworth NR (1966): Biological and phytochemical screening of plants. J Pharm Sci 55: 225–276.
- Farnsworth NR (1988): Screening plants for new medicines, in: Wilson EO, ed., Biodiversity. Washington DC, National Academy Press, pp. 83-97.
- Farnsworth NR, Soejarto DD (1985): Potential consequence of plant extinction in the United States on the current and future availability of prescription drugs. Econ Bot 39: 231–240.
- Ferreira de Lima MR, de Souza Luna J, Feitosa dos Santos A, Caño de Andrade MC, Goulart Sant’ Ana AE, Genet JP, Marquez B, Neuville L, Moreau N (2006): Anti-bacterial activity of some Brazilian medicinal plants. J Ethnopharmacol 105: 137–147.
- Finney DJ (1971): Probit Analysis, third edition. Cambridge, Cambridge University Press, 333 pp.
- González A, Ferreira F, Vázquez A, Moyna P, Paz EA (1993): Biological screening of Uruguayan medicinal plants. J Ethnopharmacol 39: 217–220.
- Greensfelder L (2000): Herbal product linked to cancer. Science 288: 1946.
- Guerrero RO, Rivera SM, Rivera S, Sueiro LA (2003): Bioassay screening of Amazonian plants. P R Health Sci J 22: 291–297.
- Hammer ML, Johns EA (1993): Tapping an Amazonian plethora: Four medicinal plants of Marajó Island, Pará (Brazil). J Ethno-pharmacol 40: 53–75.
- Harborne JB (1988): Phytochemical Methods: A Guide to Modern Techniques of Plant Analysis. New York, Chapman and Hall, 288 pp.
- Hernández NE, Tereschuk ML, Abdala LR (2000): Antimicrobial activity of flavonoids in medicinal plants from Tafi del Valle (Tucumán, Argentina). J Ethnopharmacol 73: 317–322.
- Holetz FB, Pessini GL, Sanches NR, Garcia Cortez DA, Nakamura CV, Dias Filho BP (2002): Screening of some plants used in the Brazilian folk medicine for the treatment of infectious diseases. Mem I Oswaldo Cruz 97: 1027–1031.
- Horgen FD, Edrada RA, de los Reyes G, Agcaoili F, Madulid DA, Wongpanich V, Angerhofer CK, Pezzuto JM, Soejarto DD, Farnsworth NR (2001): Biological screening of rain forest plot trees from Palawan Island (Philippines). Phytomedicine 8: 71–81.
- Lawrence GHM (1951): Taxonomy of Vascular Plants. New York, Macmillan, 823 pp.
- Lentz DL, Clark AM, Hufford CD, Meurer-Grimes B, Passreiter CM, Cordero J, Ibrahimi O, Okunade AL (1998): Antimicrobial properties of Honduran medicinal plants. J Ethnopharmacol 63: 253–263.
- Logarto Parra A, Silva Yhebra R, Guerra Sardiñas I, Iglesias Buela L (2001): Comparative study of the assay of Artemia salina L. and the estimate of the medium lethal dose (LD50 value) in mice, to determine oral acute toxicity of plant extracts. Phytomedicine 8: 395–400.
- Martinez MJ, Betancourt J, Alonso-Gonzáles N, Jaurequi A (1996): Screening of some Cuban medicinal plants for antimicrobial activity. J Ethnopharmacol 52: 171–174.
- McCuthcheon AR, Ellis SM, Hancock REW, Towers GHN (1992): Antibiotic screening of medicinal plants of the British Columbian native peoples. J Ethnopharmacol 37: 213–223.
- McLaughlin JL, Chang CJ, Smith DL (1991): “Bench-top” bioassays for the discovery of bioactive natural products: An update, in:Rahman A, ed., Studies in Natural Product Chemistry, Vol. 9. Amsterdam, Elsevier, pp. 383–409.
- Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DE, McLaughlin JL (1982): Brine shrimp: A convenient general bioassay for active plant constituents. Planta Med 45: 31–34.
- Michel J, Duarte RE, Bolton JL, Huang Y, Caceres A, Veliz M, Soejarto DD, Mahady GB (2007): Medical potential of plants used by the Q’eqchi Maya of Livingston, Guatemala for the treatment of women’s health complaints. J Ethnopharmacol 114: 92–101.
- Nick A, Rali T, Sticher O (1995): Biological screening of traditional medicinal plants from Papua New Guinea. J Ethnopharmacol 49: 147–156.
- Nortier J L, Muniz Martinez MC, Schmeiser HH, Arlt VM, Bieler CA, Petein M, Depierreux MF, De Pauw L, Abramowicz D, Vereerstraeten P, Vanherweghem JL (2000): Urothelial carcinoma associated with the use of a Chinese herb (Aristolochia fangchi). New Engl J Med 342: 1686–1692.
- Padmaja R, Arun PC, Prashanth D, Deepak M, Amit A, Anjana M (2002): Brine shrimp lethality bioassay of selected Indian medicinal plants. Fitoterapia 73: 508–510.
- Pretorius JC, Magama S, Zietsman PC (2003): Purification and identification of antibacterial compounds from Euclea crispa subsp. crispa (Ebenaceae) leaves. South African J Bot 69: 186–192.
- Quignard ELJ, Nunomura SM, Pohlit AM, Alecrim AM, Pinto ACS, Portela CN, Oliveira CP, Don LC, Silva LFR, Henrique MC, Santos M, Pinto PS, Silva SG (2004): Median lethal concentrations of Amazonian plant extracts in the brine shrimp assay. Pharm Biol 42: 253–257.
- Sánchez C, Gupta M, Vásquez M, de Noriega YM, Montenegro G (1993): Bioassay with brine Artemia to predict antibacterial and pharmacologic activity. Rev Med Panama 18: 62–69.
- Sánchez-Medina A, García-Sosa K, May-Pat F, Peña-Rodríguez LM (2001): Evaluation of biological activity of crude extracts from plants used in Yucatan traditional medicine part I. Antioxidant, antimicrobial and beta-glucosidase inhibition activities. Phyto-medicine 8: 144–151.
- Santos Pimenta LP, Pinto GB, Takahashi JA, e Silva LG, Boaventura MA (2003): Biological screening of annonaceous Brazilian medicinal plants using Artemia salina (brine shrimp test). Phytomedicine 10: 209–212.
- Smutko G (1985): La mosquitia: Historia y cultura de la Costa Atlántica. Managua, Nicaragua, Editorial La Ocarina, 179 pp.
- Soejarto DD (1993): Logistics and politics in plant drug discovery: The other end of the spectrum, in: Kinghorn AD & Baladrin MF, eds, Human Medicinal Agents from Plants. Washington DC, American Chemical Society, p. 97.
- Soejarto DD, Gyllenhaal C, Ashton PS, Sohmer SH (1996): Plant explorations in Asia under the sponsorship of the National Cancer Institute, 1986-1991: An overview, in:Balick MJ, Elisabetsky E, Laird SA, eds, Medicinal Resources of the Tropical Forests: Biodiversity and its Importance to Human Health. New York, Columbia University Press, p. 285.
- Stahl E (1969): Thin-Layer Chromatography: A Laboratory Handbook. Berlin, Springer-Verlag, 1041 pp.
- Stermitz FR, Belovsky GN, Ng D, Singer MC (1989): Quinolizidine alkaloids obtained from Lupinus fulcratus (Leguminosae) fail to influence the specialist herbivore Euphydryas editha (Lepidoptera). J Chem Ecol 15: 2521–2530.
- Stevens WD, Ulloa C, Pool A, Montiel OM (2001): Flora de Nicaragua, Vol. I-III. St. Louis, MO, Missouri Botanical Garden Press, 2666 pp.
- Stone R (2008): Lifting the veil on traditional Chinese medicine. Science 319: 709–710.
- Suffredini IB, Paciencia ML, Nepomuceno DC, Younes RN, Varella AD (2006): Antibacterial and cytotoxic activity of Brazilian plant extracts-Clusiaceae. Mem I Oswaldo Cruz 101: 287–290.
- Sutton SY (1989): Nicaragua. In:Campbell DG, Hammond HD, eds., Floristic Inventory of Tropical Countries: The Status of Plant Systematics, Collections, and Vegetation, Plus Recommend-ations for the Future. New York, New York Botanical Garden, pp. 299–304.
- Taha A, Alsayed H (2000): Brine shrimp bioassay of ethanol extracts of Sesuvium verrucosum, Salsola baryosma and Zygophyllum quatarense medicinal plants from Bahrain. Phytother Res 14: 48–50.
- Turker AU, Camper ND (2002): Biological activity of common mullein, a medicinal plant. J Ethnopharmacol 82: 117–125.
- Tyler VE, Brady LR, Robbers JE (1988): Pharmacognosy. Philadelphia, Lea & Febiger, 519 pp.
- Vila R, Iglesias J, Cañigueral S, Santana AI, Solis PN, Gupta MP (2004): Constituents and biological activity of the essential oil of Eugenia acapulcensis Steud. J Essent Oil Res 16: 384–386.
- Van Slambrouck S, Daniels AL, Hooten CJ, Brock SL, Jenkins AR, Ogasawara MA, Baker JM, Adkins G, Elias EM, Agustin VJ, Constantine SR, Pullin MJ, Shors ST, Kornienko A, Steelant WF (2007): Effects of crude aqueous medicinal plant extracts on growth and invasion of breast cancer cells. Oncol Rep 17: 487–492.
- Wanyoike GN, Chhabra SC, Lang’at-Thoruwa CC, Omar SA (2004): Brine shrimp toxicity and antiplasmodial activity of five Kenyan medicinal plants. J Ethnopharmacol 90: 129–133.
- Zakaria ZA, Khairi HM, Somchit MN, Sulaiman MR, Mat Jais AM, Reezal I, Mat Zaid NN, Abdul Wahab SNZ, Fadzil NS, Abdullah M, Fatimah CA (2006): The in vitro antibacterial activity and brine shrimp toxicity of Manihot esculenta var. Sri Pontian (Euphorbiaceae) extracts. Int J Pharmacol 2: 216–220.