Abstract
Context: Presence/absence tests for alkaloids of 31 medicinal vascular plant species from 31 genera and 26 families of eastern Nicaragua provided a baseline for bioactivity tests.
Objective: To determine the bioactivity and cytoxicity of aqueous extracts of widely used medicinal species in eastern Nicaragua.
Materials and methods: Ethnomedicinal applications were obtained from interviews of traditional healers. We used Dragendorff’s reagent to test alkaloids and brine shrimp for cytotoxicity of aqueous extracts.
Results: Twenty-nine of the 31 species tested positive for alkaloids. The median lethal concentration that kills 50% of the larvae within 24 h of contact with the extract (LC50 was less than 1000 µg/mL for 4 (13%) species (the usual cytotoxic category), 1001–5000 µg/mL for 23 (74%) species, and between 5001–7500 µg/mL for the remaining 4 (13%) species.
Discussion and Conclusion: Twenty-five of the ethnomedicines contain alkaloids but are not cytotoxic. In contrast to first suppositions, we suggest that this is a good and desirable, and perhaps expected, outcome. Medicinal plants that are cytotoxic may obviously control or kill bacteria or other pathogens, but may also negatively affect the patient; some high alkaloid levels have been associated with carcinogens. Thus, perhaps the majority of effective medicinals should be expected to be noncytotoxic. We suggest that this is a new paradigm for consideration of the overall value and effectiveness of medicinals. Of course, medicinals also can be effective in numerous ways (e.g., organ stimulation or other physiological functions) other than simply as antimicrobials or antipathogens.
Introduction
Plants are the oldest source of pharmacologically active compounds and serve as the primary source of medicine for people in developing countries, particularly in the tropics (CitationFarnsworth, 1994; CitationFarnsworth & Soejarto, 1991). In spite of this, the flora of many tropical countries is poorly documented, and more importantly, the pharmaceutical potential of most tropical species remains untested, a situation that exists in eastern Nicaragua.
Nicaragua is a nation of 140,000 km2, of which 41,700 km2 (30%) is forested with over 6000 species of vascular plants (CitationStevens et al., 2001, Citation2009; CitationSutton, 1989). In 1977, the Missouri Botanical Garden began the first systematic study of the flora and published the “Flora de Nicaragua” (CitationStevens et al., 2001, Citation2009). The Eastern Lowlands, which is the most extensive ecoregion in Nicaragua at about 33% of the country, remained botanically poorly known to modern systematics until recently (CitationTaylor, 1959, Citation1962, Citation1963; CitationSeymour, 1980; CitationDennis, 1988; CitationBoucher, 1990; CitationBoucher et al., 1990, Citation1994, Citation1996, Citation2001; CitationVandermeer et al., 1990a, Citation1990b, Citation1991, Citation1996, Citation1997, Citation1999, Citation2001; CitationYih et al., 1991; CitationBarrett, 1994; CitationCoe, 1994, Citation2008a, Citation2008b, Citation2008c; CitationPerfecto et al., 1994; CitationFerguson et al., 1995; CitationCoe & Anderson, 1996a, Citation1996b, Citation1997, Citation1999, Citation2005; CitationUrquhart, 1997; CitationGranzow et al., 1998; CitationStevens et al., 2001, Citation2009; CitationVandermeer & Granzow, 2004; CitationMascaro et al., 2005). The vascular plant flora of eastern Nicaragua is estimated at over 3000 species (CitationCoe & Anderson, 1999; CitationStevens et al., 2001). Unfortunately, this unexplored reservoir of botanical resources and its phytochemical potential are being lost due to rampant deforestation much faster than it is being surveyed (CitationLangley, 1981; CitationCoe & Anderson, 1999).
It is well documented that the screening of the ethnomedicinal flora is important in the discovery of new drugs (CitationFarnsworth, 1990; CitationCox, 1994; CitationCox & Balick, 1994; CitationKing & Tempesta, 1994; CitationCragg et al., 1997; CitationBalick & Cox, 1999). Of additional value is the standard screening of plants for the presence of bioactive compounds, and the assessment of extract toxicity via bioassays. Toxicity assays have been performed on plant extracts from the ethnomedicinal flora of several countries, including Argentina (CitationMongelli et al., 1996), Brazil (CitationQuignard et al., 2004), Guatemala (CitationFranssen et al., 1997; CitationCáceres et al., 1998; CitationMichel et al., 2007), Honduras (CitationLentz et al., 1998), India (CitationPadmaja et al., 2002), Jamaica (CitationFacey et al., 1999), New Guinea (CitationRao, 1996), Philippine Islands (CitationHorgen et al., 2001), Spain (CitationSerrano, 1996), Tanzania (CitationMoshi et al., 2004), and Turkey (CitationSener et al., 1998). A massive new and important project recently announced is the Herbalome Project by the Chinese government to assay for chemical constituents and toxicity the 400,000 medicinal preparations derived from 10,000 medicinal species used in Chinese traditional medicine (CitationStone, 2008).
Despite the importance of screening plant extracts for the discovery of new drugs, the ethnopharmacopoeia of many Mesoameriandian groups remains untapped; such is the case in eastern Nicaragua. Over the past 15 years, several studies were conducted to screen the medicinal species of eastern Nicaragua for alkaloids (CitationCoe, 1994, Citation2008a, Citation2008b, Citation2008c; CitationCoe & Anderson, 1996a, Citation1996b, Citation1997, Citation1999, Citation2005). However, bioassays to determine the toxicity of these medicinal species were not conducted until recently (CitationCoe et al., 2010). This study reports on measures of potential effectiveness and cytotoxicity of plant extracts derived from medicinals used by traditional healers in eastern Nicaragua. In what follows, we report the results of alkaloid tests and brine shrimp bioassays of medicinal plant aqueous extracts of 31 species currently used by healers in eastern Nicaragua. We develop the arguments as well that cytotoxicity levels, high and low, are of considerable interest. High levels offer insights into effectiveness in treating such things as bacteria or protozoan-based illnesses. But some high cytotoxic levels may be associated with detrimental effects on patients as well. Thus, either way, cytotoxicity tests are of importance.
Materials and methods
Plant collection
Plant material was collected during field studies by one of us (Coe) working with traditional healers (shamans, midwives, and herbalists) during several months over many years (1992–2008). Further details about the ethnobotanical studies are published elsewhere (CitationCoe, 1994, Citation2008a, Citation2008b, Citation2008c; CitationCoe & Anderson, 1996a, Citation2008b, Citation1997, Citation1999, Citation2005). Voucher specimens were deposited at the Missouri Botanical Garden (MO, USA) and the University of Connecticut (CONN, USA). Vouchers were identified by Coe with the assistance of specialists listed in the acknowledgments.
Alkaloid screening
Thirty-one medicinal species were screened for alkaloids (). Plant parts tested are the same as those used in the preparation of herbal remedies, and included both aerial and underground parts as appropriate (). 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 using Dragendorff’s reagent (CitationHarborne, 1988; CitationStermitz et al., 1989) and thin-layer chromatography (TLC) (CitationStahl, 1969; CitationStermitz et al., 1989). 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, 0.5–1.0 mL of 2:1 CHCl3-MeOH was added. The mixture was then mixed with a stirring rod for 3–5 min, and afterward allowed to stand and separate into two phases (upper and lower). The lower phase containing the plant extract dissolved in the CHCl3 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 amount was spotted on an aluminum-backed TLC 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 when at least two of three replicates gave positive results. We are aware that these kinds of alkaloid tests can sometimes produce false-positive reactions, especially in latex-bearing families, e.g., Apocynaceae, Araceae, Clusiaceae, Convolvulaceae, and Moraceae (CitationFarnsworth, 1966). However, the method (CitationStermitz et al., 1989) we used includes a purification procedure (adding a base-Na2CO3 and extraction with a water immiscible organic solvent-CHCl3) that helps avoid false-positive reactions. But, given the doubt, as an additional measure, we verified our test results, for the five latex-bearing families with reports in the literature.
Table 1. Bioactivity and bioassay results of medicinal plants used in eastern Nicaragua.
Plant crude extract preparation
We made every attempt here to follow the general preparation procedure that would be utilized by field medicinal practitioners. This included, especially, using the same plant parts as employed in the field, and extracting in water− the most common mode of preparation of medicinals (CitationCoe, 1994, Citation2008a, Citation2008b, Citation2008c; CitationCoe & Anderson, 1996a, Citation2008b, Citation1997, Citation1999, Citation2005). 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); this was the stock solution. An appropriate amount of 1% NaCl solution was added to the stock solution to give concentrations of 500, 1000, 2500, 5000, 7500, and 10,000 µg/mL. 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. The “Control Solution” we used consisted of only 1% NaCl solution.
Hatching of brine shrimp
Eggs of brine shrimp (Artemia salina L., Artemiidae) were purchased from Carolina Biological Supply (Burlington, NC, USA) and were incubated for 48 h in a culture vessel (15 × 15 × 15 cm) containing 1% NaCl, the latter, prepared from nitrate, phosphate, and silicate-free sea salt and deionized water (35 g/L) at 24°C to 28°C under a lamp. The saltwater solution was aerated continuously during incubation with an aquarium air pump. 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; CitationCoe et al., 2010). 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 death 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 U.S. Environmental Protection Agency, Cincinnati, Ohio, from which median lethal concentrations (LC50) at 95% confidence intervals (CI) were calculated, according to the method of CitationFinney (1971). Biological activity using the BSLA was recorded as a lethal concentration (LC50) when 50% of the larvae were killed within 24 h of contact with the extract. As is standard practice (CitationMeyer et al., 1982; CitationZhao et al., 1999; CitationMorrow et al., 2001; CitationHoletz et al., 2002; CitationPimentel Montanher et al., 2002; CitationSilva et al., 2007) LC50 values lower than or equal to 1000 µg/mL were considered cytotoxic and greater than 1000 µg/mL for plant extracts were considered noncytotoxic.
Results
The 31 species assayed constitute a diversity of plants as illustrated by their distribution among 31 genera and 26 families, including two fern and 29 angiosperm species. The ethnomedicinal uses, bioactivity, and bioassay information are presented in and . Almost 95% (29/31) of the species screened for alkaloids tested positive (). However, in the BSLAs, only four species (13%) were cytotoxic to brine shrimp at 1000 µg/mL () or less – a level that is generally considered cytotoxic (CitationMeyer et al., 1982; CitationZhao et al., 1999; CitationMorrow et al., 2001; CitationHoletz et al., 2002; CitationPimentel Montanher et al., 2002; CitationSilva et al., 2007). All four of these species tested positive for alkaloids. Almost 75% of the species (23) had an LC50 at between 1000 and 5000 µg/mL and the remaining four required a concentration greater than 5000 µg/mL to be cytotoxic. The two species that did not test positive for alkaloids were in the 1000–5000 µg/mL group. Given that 27 of the species were cytotoxic at levels above the 1000 µg/mL threshold, the vast majority of the species tested with the bioassays should be considered “non-cytotoxic.”
Table 2. Medicinal applications and the number of species used of the 31 medicinal assayed.
The four species with cytotoxic extracts were distributed across a wide range of plants – and included a fern (see ). In addition to alkaloids, the four cytotoxic species have been tested in other contexts, and apparently contain other secondary metabolites such as fatty acids, flavanoids, glycosides, phenols, saponins, tannins, and terpenoids (CitationGibbs, 1974; CitationCambie & Ash, 1994; CitationHess et al., 1995; CitationCalderon et al., 2000, Citation2001; CitationZucaro et al., 2000). Obviously, these other compounds might have been the source of the cytotoxicity as well as, or instead, of the alkaloids.
Discussion
People in eastern Nicaragua use a wide range of plants/plant extracts for treatment of an array of ailments (CitationCoe, 1994, Citation2008a, Citation2008b, Citation2008c; CitationCoe & Anderson, 1996a, Citation1996b, Citation1997, Citation1999, Citation2005). One of the authors (Coe) has studied the plants in the field and there are several papers citing medicinal applications (CitationCoe, 1994, Citation2008a, Citation2008b, Citation2008c; CitationCoe & Anderson, 1996a, Citation1996b, Citation1997, Citation1999, Citation2005). In spite of the fact that the 31 species tested in this study are widely used by healers in eastern Nicaragua to treat over 25 ailments (see and ), there is virtually nothing known of the cytotoxicity. The recent discovery of the carcinogenic effects of the extracts of Aristolochia fangchi Y.C.Wu ex L.D.Chow & S.M.Hwang (Aristolochiaceae), a medicinal species widely used in Asia, highlights the importance of toxicity screening (CitationGreensfelder, 2000; CitationNortier et al., 2000) of medicinals. Toxicity can be good – potentially killing bacteria and parasites. And/or it can be bad, as with A. fangchi, negatively impacting the patient – perhaps in ways much worse than the ailments treated. Many species of the genus Aristolochia are commonly used in eastern Nicaragua and elsewhere to treat a variety of ailments, from skin rashes to snakebites (CitationMorton, 1981; CitationCoe, 1994, Citation2008a, Citation2008b, Citation2008c; CitationCoe & Anderson, 1996a, Citation1996b, Citation1997, Citation1999, Citation2005). Thus, Aristolochia-bearing medicinals in Nicaragua may be carcinogenic as well. We did not have the Aristolochia (that has been a part of our other studies – CitationCoe & Anderson, 1996a, Citation1996b, Citation1997, Citation1999, Citation2005; CitationCoe, 2008a, Citation2008b, Citation2008c) to test, but the same idea applies to the other medicinals used: any of the eastern Nicaraguan medicinals may be cytotoxic.
In our studies, less than 10% (4/31) was considered cytotoxic in spite of the fact that over 90% (29/31) of the species contained alkaloids (). It is possible that the type of alkaloid in the extract is not toxic to the test organism, or that the solvent used in the alkaloid extraction (which was distilled water – to match the most commonly used method of preparation in eastern Nicaragua) was not effective in removing all the alkaloids from the plant tissue. Thus, we can conclude that our results clearly suggest that the presence of the water-extracted alkaloids do not necessarily mean that the plant extract will be cytotoxic. The lack of cytotoxicity, of course, does not mean the medicinal is not effective (see more on that below).
Similarly, the two species (Crescentia cujete L., Bignoniaceae; Melicoccus bijugatus Jacq., Sapindaceae) that tested negative for alkaloids are fairly widely used medicinals (). The effectiveness of these two species as herbal remedies may be due to the presence of other types of secondary metabolites such as flavanoids, glycosides, phenols, sapogenins, saponins, and tannins (CitationHeltzel et al., 1993a, Citation1993b; CitationBinutu & Lajubutu, 1994; CitationKaneko et al., 1997, Citation1998; CitationBetancur-Galvis et al., 1999; CitationBystrom et al., 2008; CitationOgbuagu, 2008). For example, the fruit pulp of C. cujete contains flavanoids, saponins, cyanogenic glycosides (as HCN), phenols, and tannins (CitationOgbuagu, 2008). Similarly, the fruit pulp of M. bijugatus has phenolic acids, flavonoids, procyanidins, and catechins (CitationBystrom et al., 2008). The therapeutic values and toxicity of these other compounds are well known.
Cymbopogon citratus Stapf. (Poaceae) is also interesting. This species tested positive for alkaloids, but is also reputed to contain a wide array of other secondary metabolites (CitationCambie & Ash, 1994). In spite of the alkaloids and secondary metabolites, the test for this species put it into the category of “noncytotoxic.” Interestingly, ethanol extracts of C. citratus also did not demonstrate any significant biological activity – in this case, antibacterial effect against Staphyloccocus aureus, S. epidermidis or to methicillin-resistant S. aureus (MRSA) (CitationChomnawang et al., 2009). Thus, the extracts from this species are probably not effective cytotoxic medicinals, but rather have an effect on other physiological functions.
In our study, four extracts widely used by many Amerindians to treat a variety of ailments such as inflammation, mouth and skin sores, fever, muscular pain, rheumatism, respiratory disorders, and pulmonary congestion showed the greatest cytotoxicity. The fern species, Lygodium heterodoxum Kunze (Schizaeaceae) is particularly interesting because ferns in general are not known for their bioactive compounds (CitationBerti & Bottari, 1968). However, in our study, this fern had the highest cytotoxicity, causing brine shrimp death at slightly above 700 µg/mL (). The effectiveness of L. heterodoxum at this relatively low concentration may be due to the presence of the alkaloids, or other secondary metabolites such as flavanoids, glycosides, phenols, saponins, and tannins (CitationGibbs, 1974) that it contains. The additional three species that were cytotoxic also contained alkaloids (), but, like the L. heterodoxum, they posses other secondary metabolites as well (CitationGibbs, 1974; CitationHess et al., 1995; CitationCalderon et al., 2000). For instance, Eugenia venezuelensis O. Berg. (Myrtaceae) contain coumarins (such as 3,4- benzocoumarin), glycosides, phenols (such as d-quercitol), eugenol, eugenol-acetylsalicylic acid ester, methyl salicylate, monoterpenoids, triterpenoids (such as oleanolic acid), saponins, and tannins (CitationGibbs, 1974; CitationCambie & Ash, 1994). Olyra latifolia L. (Poaceae) contains carbohydrates such as fructosans, phenolic acids such as caffeic, ferulic acid, gentisic acid, p-OH-benzoic acid, flavonoids such as anthocyanins, leucoanthocyanins, flavones, and flavonols, p-coumaric acid (CitationGibbs, 1974). Vochysia ferruginea Mart. (Vochysiaceae) contains fatty acids such as lauric acid, myristic acid, tannins, triterpene such as 6β-hydroxymaslinic acid, polyphenols such as β-sitosterol-glucoside, uvaol, erythrodiol, urosolic acid, oleanolic acid, crategolic acid, bellericagenin A, bellericaside A, betulinic acid (CitationGibbs, 1974; CitationZucaro et al., 2000). It is clear, in particular, that V. ferruginea contains a number of cytotoxic compounds, and, thus, not surprisingly, showed bioactivity (e.g., anaesthetic, analgesic, antifungal, antiseptic, bactericide, and anti-inflammatory activities) in many other assays (CitationGibbs, 1974; CitationCambie & Ash, 1994; CitationHess et al., 1995; CitationCalderon et al., 2000, Citation2001; CitationZucaro et al., 2000). However, in general, it is obvious that all four of the species we found to be most lethal are loaded with compounds that may have caused the cytotoxicity.
Among the 26 ailments/aliment categories that are included in , there are three that are obviously associated with external bacterial, protozoan, etc., vectors: “Infections” (10 species), “Malaria” (1 species), and “Worms and intestinal parasites” (1 species). We scored the species that are used to treat these vector-borne ailments, and compared their distribution among the three LC50 categories (). The patterns revealed are interesting. Among the four “effective” species (those that had an effect in the bioassay at less than 1000 μg/mL), three are not used to treat the vector-borne ailments. That result argues against the concept that a strong bioassay effect (“cytotoxicity” as here used) is a key to application in treating pathogen-borne ailments. Slightly more than half of the 23 species (13) in the next category, 1000–5000 μg/mL, also were not used to treat pathogens, but the difference between those “used” and “not used” is not significant (chi-square goodness of fit, P = 0.53 = NS). Thus, this result is thus “neutral” in arguing for or against medicinal application to vector-borne ailments. The only two medicinal species that lacked alkaloids were in the “not used” category. Finally, none of the four species in the “weakest” category (i.e. more than 5000 μg/mL required for an effect in the bioassays) were used to treat vector-borne ailments, a result one might expect if cytotoxicity bioassays were a good measure of application to vector-borne diseases. Overall, the variable and somewhat contradictory results from this analysis reinforce significant conclusions from our study: that the major application of the native medicinals (alkaloid-bearing or not) is (a) not necessarily in the treatment of infectious pathogens, and (b) that cytotoxicity is not a predictor of such an application.
Table 3. Bioactivity and use of medicinal in treatment of “Infections” (I), “Malaria” (M) and “Worms and intestinal parasites” (P).
Cytotoxicity could be considered desirable – in that bacteria and other pathogens or parasites might respond like the brine shrimp did. On the other hand, cytotoxicity might be considered undesirable, because, as for Aristolochia fangchi, perhaps the alkaloids and cocktail of other compounds might be so strong, so concentrated, that they may kill “host” cells or may be carcinogenic; the treatment might be more detrimental than the illness. Accordingly, the question is: are these cytotoxic species sufficiently valuable medicinally to be used anyway, or should they be considered dangerous – as in the case with A. fangchi?
This leads to a perhaps unexpected conclusion: the 25 alkaloid bearing, but noncytotoxic species might be, in fact, the safer and more generally applicable medicinals. These 25 noncytotoxic species are also widely used medicinals. We presume that they have some physiological and therapeutical effects. In spite of, or because of, their lack of cytotoxicity, they may or can play a role in other ways that have salutary medicinal applications (e.g., organ stimulation, increase flow of gastric juices, etc.). Perhaps, then the better approach might be to encourage the use of plant extracts that are proven noncytotoxic in tests like the ones we performed, and encourage avoidance, or limited use of the plant extracts that are cytotoxic. These results and this conceptual approach may lead us to reconsider what constitutes a desirable native medicinal.
Acknowledgments
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 Atlántica) 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, USA), Ronald Leisner (MO, USA), Amy Pool (MO, USA), Warren D. Stevens (MO, USA), and Charlotte M. Taylor (MO, USA). Thanks to Irsa Amin and Kellyn Misset for help in the laboratory, and Allison Robinson and Evita Vigante 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.
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