Ethnolichenology, Biological Activity, and Biochemistry of Amazonian Lichen Species
Azenha1, G., T. Iturriaga2, F.I. Michelangeli3, and E. Rodriguez1
(1Cornell University, 2Universidad Simon Bolívar, 3IVIC; research conducted at the Yutajé research station and IVIC, Venezuela 1997)
Lichens (a type of fungi that live in an obligate symbiosis with algae )posses a plethora of unique secondary compounds found only within this taxonomic group. Lichen compounds are known to be clinically effective antibiotics. General medications against pathogenic microorganisms that contain lichen compounds have been developed. Nonetheless, research into the potential use of lichen compounds has dwindled. This is unfortunate especially in light of research which has demonstrated that numerous lichen secondary compounds exhibit antitumor activity. The biochemistry of tropical lichens is less well known than that of temperate species. They provide a potential untapped source for novel compounds. Four species of lichen, Usnea sp. P. Browne ex Adans (Lecanorales, Usneaceae), Cladonia sp. (Hill.) (Lecanorales, Cladoniaceae), Parmelia sp. Ach. (Lecanorales, Parmeliaceae), and Cora pavonia (Web.) (Aphyllophorales, Dictyonemataceae), were collected in the vicinity of the Yutajé research station in the Amazonas State, Venezuela and extracted in ethanol, benzene, and acetone. Bacterial bioassays were conducted and Usnea sp., Cladonia sp., and Parmelia sp.were found to exhibit antibacterial activity against Staphylococcus aureus. The extracts were subject to Thin Layer Chromatographic (TLC) analysis along with acid hydrolysis tests to determine the general classes of chemical compounds present in the extracts. In addition, for Parmelia sp., ethnolichenological data was collected, concerning the employment as a medicinal to treat various afflictions, from two distinct ethnic groups, the Piaroa and the Guahibo.
Lichens produce an array of secondary compounds (primarily phenolics), many of which are absent in plant species. Over 500 primary and secondary compounds have already been encountered from over 5,000 different species of lichenized fungi (Lawrey 1984). In fact, it is one of the best studied taxonomic groups of organisms in terms of secondary chemistry. Of the over 500 compounds identified thus far over 100 are unique to lichens. Despite the wealth of knowledge of lichen chemistry, current understandings are mainly limited to structures, biogenic pathways, and phylogenetic significance of temperate lichens (Lawrey 1986).
Little research has been conducted on the secondary chemistry of tropical lichens. One would expect that tropical lichen species produce similar compounds to those produced by lichens in temperate regions, but due to the unique selection pressures placed on species in a tropical rainforest environment, other compounds that are absent in temperate species may also be expected to be found.
Following the similar logic in theories of plant secondary chemistry, lichen compounds are assumed to have an adaptive function. The energetic cost of producing these compounds leads us to believe that if they did not confer some adaptive advantage they would be selected against over evolutionary time. Lichens have a low growth rate and productivity and thus it would seem unlikely that they would waste energy on compounds of little or no adaptive value (Lawrey 1984). Current theories on their possible function include antimicrobial, alleopathic, antiherbivore, phycobiant regulation, and light-screening roles. Unfortunately our comprehension of the biological and ecological roles of these various compounds is limited (Lawrey 1986).
Lichen species have been employed as medicinals around the world. One of the best studied regions in relation to medicinal lichen use is India. Little research is available on medicinal ethnolichenology. In general, the medicinal use of fungi has been a neglected field of study compared to medicinal ethnobotany.
Lichen substances are known to be clinically effective antibiotics and many in vitro experiments with lichen extracts have demonstrated antibiotic effects (Lawrey 1986). Lichen compounds are effective antibiotics against gram positive bacteria, various fungi, and a variety of other microorganisms. Over 50 % of tested lichen compounds possess antibiotic properties (Vartia 1973).
Certain species of lichen have also been found to possess some antitumor activity (Lawrey 1986). Cetraria sp. has been employed as a "cancer" treatment for probably thousands of years (Hartwell, 1971). From the experiments done thus far on the potential antitumor activity of lichen compounds, the polysaccharide component of lichens, like psoromic and usnic acids, has been shown to be effective against tumors (Lawrey 1984). Species of Parmelia have been reportedly used as folk remedies against cancer and/or tumors in India and Chile. Several species of Usnea have been reported as a similar folk remedy in Argentina (Hartwell 1971).
Considering the established biological activity of lichen extracts and compounds and the uniqueness of lichen chemical composition, lichens have a strong potential as sources for novel compounds of medicinal value. Studies on the chemistry and biological activity of tropical lichens is sorely needed. Coupling these types of studies with ethnobiological inquiry can facilitate the search for medicinal compounds, by taping into the wealth of knowledge that rural mestizos and indigenous people posses about the environment and the use of resources found within that environment.
With this background in mind, the chemistry, biological activity, and local medicinal use of 4 species of lichens from the northern Amazonas State in southern Venezuela were studied.
Fig. 2. Cora pavonia thalli are commonly found growing on trees in varzea and terra firme forests.
Photo by Gustavo Azenha '98
Fig. 3. Researchers converse with Marco Antonio Silva about local remedies used by the Guahibo for a variety of ailments, including the use of Parmelia sp. as a wound treatment.
Photo by Natalie Emlen
MATERIALS AND METHODS
Collection and Extraction
Four species of lichen were collected in the vicinity of Yutajé in the Amazonas state of Venezuela and then extracted in order to conduct studies on the biological activity and the chemical composition of lichens. Whole lichen thalli of Cladonia sp., Cora pavonia (figure 2), Usnea sp., and Parmelia sp.(figure 1) were collected and substrate material was carefully removed. The thalli of Cladonia sp., Usnea sp., and Parmelia sp. were collected at an elevation of 200-400 meters above sea level where they were all fairly abundant. Cora pavonia thalli were collected in humid forests (at approximately 100 meters above sea level) growing on live tree trunks. Fresh material, for all collected lichen species, was macerated and extracted in 70% ethanol and warm acetone (50°). A portion of each collection was dried, macerated, and then extracted in benzene. After approximately 48 hours vacuum filtration was conducted with all extracts. The extract was then concentrated in a rotary evaporator.
Ethnolichenological Information Gathering
Data was collected on the use and nomencalature of these lichens among Piaroa and Guahibo indigenous communities. The Piaroa are a forest people inhabiting the Amazonas State, Venezuela, which live an agricultural lifestyle characterized primarily by manioc (Manihot esculenta) and corn (Zea mays) cultivation (Overing and Kaplan 1983). They represent about 21% (9,348 people) of the indigenous population of the state. The Guahibo are also an agricultural group of people inhabiting savannas in roughly the same region as the Piaroa (Metzger et al. 1983). Their population is also approximately 21% of the indigenous population of the state. They originally inhabited the Vichada region of Colombia, but have migrated to Venezuela since European colonization of the area.
Informants were shown freshly collected lichen thalli and asked whether or not the species were employed for medicinal, alimentary, or other purposes. If the species was said to be used medicinally, further inquiry was made into the specific use, collection, and preparation. Informants were also asked about the local names used for the medicinally employed lichens.
In order to confirm their identification skills, informants were then asked to help collect some fresh lichen. This served to confirm that the lichen sample used during the interviewing process was the same lichen that collaborators were providing information about. Although not specifically asked, several informants provided information about the seasonal patterns of abundance for the lichen species.
All the informants that collaborated on this project were not considered "curanderos" or "curanderas", although a couple of them are locally known to posses a superior knowledge of the medicinal plants/fungi of the region. Therefore the information collected represents knowledge that is common or fairly common among the members of the communities studied.
TLC analysis was conducted at Cornell University. For each extract, TLC plates were developed for approximately 45-55 minutes under controlled conditions in three standard elution systems. Chromatograms were developed in Brinkman tanks to a height of 10 cm on Silica Gel plates. Spots were applied 2 cm from the bottom of the plate and spaced at intervals of 1 cm. These solvents systems were benzene-dioxane-acetic acid (180:45:5, 230 ml); hexane-diethyl ether-formic acid (130:80:20, 230 ml); and toluene-acedic acid (200:30, 230 ml). Plates were visualized in short and long wave UV light. RF values were measured for all spots visualized on the plates and were subsequently compared to standard RF values from Culberson (1972).
Acid hydrolysis tests for esters and phenolic acids were conducted by spraying plates with 10% H2SO4 followed by heating to 110o for 15-30 minutes. Specific color reactions were observed under this treatment, which are correlated with the chemical structure of compounds. The color reactions observed were compared with the standard data for lichen acid hydrolysis tests found in Culberson (1972).
Bioassays were conducted at IVIC (Instituto Venezuelano de Investigaciones Científicas, Caracas, Venezuela) on several species of bacteria to determine biological activity. Those bacteria tested include Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Serratia mar., Salmonella sp., Salmonella thypi., Shigella flexneri , and Shigella sp. The bacterial strains were suspended in 5 ml of nutrient broth.
Assays were conducted using a disc-diffusion method. One hundred ul of the microorganism culture was spread on a sterile agar plate. Sterile paper discs were impregnated with 20 ul of the lichen extracts. For each of the four lichen species collected, assays were conducted with the acetone, ethanol, and benzene extracts. The extract impregnated discs were placed on the bacterial plates and maintained at room temperature.
Plates were analyzed and measured for inhibition at 24 hours and at 48 hours after the addition of the disc.
The only lichen species found to be employed by people in local communities was Parmelia sp. According to Piaroa collaborators, the lichen thallus is boiled into a tea that is drunk 3-4 times a day for a week in order to treat gonorrhea or "painful urination". It is called "odoche jupacua" which means "iguana's toe" in Piaroa. According to Guahibo informants they use the same species for different purposes: the thallus may also be boiled in water and then applied to insect bites or cuts and wounds.
During investigations into the use of lichen species as folk remedies, one informant, a "criolla" woman, indicated that a man had used an anticancer remedy that may have been the species of Parmelia studied in this project. However, her identification was tenuous and she was uncertain if Parmelia sp. was the source of the remedy used.
The results of the bacterial bioassays demonstrate activity against Staphylococcus aureus for Cladonia sp., Parmelia sp., and Usnea sp. For all other bacteria tested there was no activity. The extracts of Usnea sp. demonstrated the highest degree of inhibition of bacterial growth.
The preliminary chemical results from the TLC analysis conducted indicates a variety of compounds present in all four lichen species studied. The focus of the chromatographic analysis was on Parmelia sp. and Usnea sp. since these two species exhibited the greatest degree of bacterial inhibition.
The acid hydrolysis tests indicate the general classes of compounds present in the lichen species. For Usnea sp. the presence of fatty acids, alicyclics, orcinol depsidones, and beta-orcinol depsidones was detected. For Parmelia sp. beta-orcinol depsidones, fatty acids, and alicyclics were detected
The wide range of compounds and compound classes detected in the lichen extracts makes it difficult to determine which compounds might account for their biological activity without further investigation. The activity exhibited by the Parmelia sp. extracts against Staphylococcus aureus demonstrates the efficacy of its use by the Guahibo as a wound remedy since S. aureus is one of the most common types of bacteria to infect wounds. Wound treatments are an important remedy since pharmaceutical antibiotics are not readily available in the remote region where this study was conducted and there exists a high probability of wound infections in tropical, moist climates.
The most effective known antibacterial compounds are usnic acids, pulvinic
acid derivatives, aliphatic acids, orc
inol-type depsides, and depsidones. Usnic acid has been found to have inhibitory effects on Mycobacterium tuberculosis, the cause of tuberculosis (Vartia 1973) and Staphylococcus aureus (Asahina et al. 1954).
Cora pavonia was not found to have any activity against any of the bacteria tested. Approximately 98% of lichen species are lichenize Ascomycetes (Kendrick 1992). Therefore, most of the information regarding lichen compounds pertains to lichenized fungi in the class Ascomycetes. Cora pavonia is one of about 20 Basidiomycete lichens.
Since secondary chemistry may often exhibit phylogenetic relationships we would expect to find different chemistry and biological activity in Cora pavonia, compared to the other 3 species studied which are all lichen Ascomycetes and are also found in the same order (Lecanorales), this may account for the lack of activity against any of the bacteria.
Further investigation into the taxonomy, chemistry, and biological activity of the four lichen species is planned. It is, however, essential that the lichens are identified to species, before any further chemical and bioassay research is conducted.
More TLC analysis, in conjunction with spray tests, is planned in order to ascertain the chemical composition of known lichen compounds, and to determine if previously undescribed compounds may be present.
More testing on the biological activity of the lichen extracts is planned.
Of particular interest is the completion of
assays for antitumor activity. In addition, bacterial bioassays are planned to determine the efficacy of the lichen species against the bacteria that causes gonorrhea. Bacterial overlay TLCs are also planned with Staphylococcus aureus. These types of assays will allow determination of which particular chemicals within the lichen extract account for its antibacterial activity.
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Culberson, C. F., and H. Kristinsson. 1970. A standardized method for the identification of lichen products. Journal of Chromatography, 46:85-93.
Culberson, W. L., and C. F. Culberson. 1970. A phylogenetic view of chemical evolution in lichens. The Bryologist, 73(1): 1-31.
Hartwell. 1971. Plants used against cancer. Lloyodia, 34(4): 414-416
Kendrick, B. 1992. Lichens: dual organisms. The Fifth Kingdom. Newburyport, MA: Mycologue Publications. 114-119.
Lawrey, J. D. 1986. Biological role of lichen substances. The Bryologist, 89(2): 111-122.
Lawrey, J. D. 1984. Lichen secondary chemistry and lichen secondary chemical ecology. In Biology of Lichenized Fungi. New York; Praeger. 193-263.
Metzger, D. J., and R. V. Morey. 1983. Los Hiwi (Guahibo). Los Aborigenes de Venezuela. Caracas. 125-216.
Overing, J., and M. R. Kaplan. 1983. Los Wothuha (Piaroa). Los Aborigenes de Venezuela. Caracas. 307-412.
Vartia, K. O. 1973. Antibiotics in lichens. In V. Ahmadjian & M. E. Hale, Jr. (eds.),The Lichens. New York: Academic Press. pp 547-561.
I would like to thank Dr. Stanford Zent, and Eglee Zent (IVIC), our Guahibo collaborators Marco Antonio Silva and Angelica Fernandez García, and our Piaroa collaborators Basilio Moreno and Julio Moreno. In addition I would like to thank MIRT-NIH for their generous financial support.
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