Emanations Volume 4

ABSTRACT

The most common and one of the most easily observable of Amazonian primates, the Common Squirrel Monkey (Saimiri sciureus), was studied in the Yarapa River area (a varzea forest region in Loreto, Peru) in order to investigate the chemical ecology and possible medicinal value of plants common to their diet. In a study area of approx. 20 km2, data was collected on the behaviors and diet of the locally abundant S. sciureus through direct observation and interviews with indigenous people of the area. Four fruits eaten by the monkeys were analyzed-Paullinia alata (Sapindaceae), Cecropia membranacea (Cecropiaceae), Gustavia longifolia (Lecythidaceae), and Schinopsis peruviana (Anacardiaceae). In order to determine if these fruits are ingested by S. sciureus for medicinal or purely nutritional purposes, a preliminary survey of their chemistry was initiated. Leaf and fruit extracts were tested for bioactivity against the bacteria Bacillus cereus (gram-positive) and Pseudomonas aeruginosa (gram-negative) and the yeast Candida albicans. In addition, a brine shrimp assay was used to assess the cytotoxicity of the extracts. Thin-layer chromatography (TLC) was performed on all extracts, and the free radical agent, 2,2-diphenyl-1-picrylhydrazyl (DPPH) was used to confirm that multiple antioxidant compounds are present in extracts of all fruits and seeds. No anti-fungal activity was found in any of the four species studied. However, the leaves of the C. membranacea and the leaves and fruit of the S. peruviana exhibited anti-bacterial activity (gram positive and negative). All of the fruit extracts (both the whole fruits and the pulp and seed extracts) showed possible cytotoxic activity. Further field investigation should be performed to clarify if the bioactive compounds of the fruit ingested by S. sciureus are beneficial or harmful, or if the fruits are ingested purely for nutritional value.

The difficulties in observing the new world primates, along with the lack of popular support for studying primates which are not as ‘human-like’ as some old world primates, has led to a poor understanding of their diet and chemical ecology. One of the most often encountered primates in the Neotropics, the Common Squirrel Monkey (Saimiri sciureus), was chosen as the focus of study in the varzea forest region along the Yarapa River in Peru. Although other Neotropical primates are more sedentary, living in the same tree for weeks or possibly years (and consequently being easier to maintain a long term study of the same individuals), S. sciureus has a large range and was observed to forage regularly in groups of 30 to 50 members, making it an ideal species for a dietary study. The focus of this study is the determination of the chemical basis of the primates’ choice of particular plants in its diet. Previous studies of Neotropical primates, such as capuchins (Cebus sp.) have indicated the use of plants for medical purposes (Baker 1996). The possibility of similar zoopharmacognostic relationships increases in regions of high biodiversity in which the animal subject has a highly varied diet.

The fruits the following plants were observed to be eaten by S. sciureus and were subsequently extracted and analyzed: Paullinia alata (Sapindaceae), Cecropia membranacea (Cecropiaceae), Gustavia longifolia (Lecythidaceae), and Schinopsis peruviana (Anacardiaceae). While S. sciureus was not seen to eat leaves, many other Neotropical primates are known to do so; therefore the leaves of these plants were also examined. The fruit of C. membranacea and S. peruviana are eaten whole by S. sciureus, so the extractions were made of whole fruit. Since the primates only eat the pulp of the other two fruits (P. alata and G. longifolia), separate extractions were made of pulp and seeds. Bioassays were then used to determine whether the plants have been selected by the primates for purely nutritional reasons or whether secondary metabolites play a role in attracting or dissuading them from eating the fruit.

MATERIALS AND METHODS
Field Study

In a study area of approximately 20 km2, several troops of S. sciureus were observed for their diet and behavior. Local informants with first-hand knowledge of the S. sciureus diet were interviewed as well.

Collection and Extraction

For C. membranacea and S. peruviana, extractions were made of the entire fruit in a 3:1 ratio of 96% ethanol to water, and leaves in 96% ethanol. For P. alata and G. longifolia, three separate extractions were made: pulp (3:1, 96% ethanol: water), seeds (3:1, 96% ethanol: water), and leaves (96% ethanol). Leaves were chopped into pieces, and whole fruit, pulp, and seeds were crushed before being extracted for at least 24 hours.

Anti-oxidant Assay

Thin-layer chromatography (TLC) was conducted on aluminum backed plates coated with silica gel and exposed to the free radical reagent 2,2-diphenyl-1-picrylhydrazyl (DPPH) to visualize anti-oxidant activity. A yellow spot showing neutralization of the dark purple free radical indicated the presence of anti-oxidant compounds. Samples were then hydrolyzed by a 1:10 ratio of HCl: extract. TLC was then performed on the hydrolyzed extracts, and the anti-oxidant assay was redone to determine the effect of hydrolysis on the activity of the compounds.

Anti-microbial Bioassay

Anti-fungal and anti-bacterial activity was measured using a qualitative disk diffusion assay. The organisms used were the gram-positive bacteria Bacillus cereus (ATCC # 11778), gram-negative bacteria Pseudomonas aeruginosa (ATCC # 27853), and the yeast Candida albicans (ATCC # 90028). Sterile disks were impregnated with 20l of extracts of unknown concentration, placed on inoculated plates, and incubated at 37oC for 24 hours. Anti-bacterial assays were conducted on nutrient agar (Difco 0001) and anti-fungal assays on Sabouraud Dextrose Broth (Difco 0382).

Cytotoxicity Bioassay

Three drops of extract were added to small vials of approximately 30 brine shrimp and the shrimp were observed after 6 and 24 hours. Two control groups, one with only food added and another with food and three drops of 96% ethanol, were also observed at the same time intervals. Cytotoxicity was measured by the percentage of brine shrimp killed after 6 and 24 hours.

RESULTS AND DISCUSSION

The high variety of the S. sciureus diet prohibits a comprehensive understanding of all of the chemical and ecological interactions in this preliminary study.

The results of the bioassays are qualitative, but nonetheless informative. The C. membranacea leaves and the S. peruviana fruit and leaves, which exhibited a greater than 2mm zone of inhibition for both gram-negative and gram-positive bacteria, were the only extracts to show any bioactivity. The anti-oxidant assay, which was conducted on TLC plates after separation, revealed anti-oxidant compounds in each extract. Many of the extracts contained multiple active compounds. This anti-oxidant activity was particularly strong in the P. alata pulp and G. longifolia seeds. After hydrolysis of the samples, some of the compounds which were previously anti-oxidant had lost their activity-showing that some of the active compounds contain ether or ester bonds required for activity. This could be due to an anti-oxidant glycoside that is inactive as an aglycone. The cytotoxicity assay showed nearly all extracts to be cytotoxic. However, the fact that the control groups had death rates of 83 % and 80 % calls into question the validity of the results. Thus, further controlled cytotoxicity assays should be conducted before conclusions can be drawn from the data.

Research has been done on the Cecropia and Schinopsis genus, and on Paullinia cupana, or guarana, a close relative of P. alata. A previous study of Schinopsis balansae shows mild anti-microbial activity (Salvat 2001), agreeing with the data of this study. A study of Cecropia obtusifolia showed analgesic as well as anti-inflammatory activity (Perez-Guerrero et al 2001). Research on P. cupana has shown caffeine, theophylline (Belliardo et al 1985), inhibition of platelet aggregation (Bydlowsky et al 1988), anti-oxidant activity (Mattei et al 1998), and increased physical and mental capacity (Espinola et al 1997), as well as a low toxicity (Espinola et al 1997, Mattei et al 1998, Santa Maria et al 1998). Most of the fruits studied here are unlikely to be harmful to S. sciureus, because their seeds are primarily dispersed by the monkey. G. longifolia is an exception in which the relationship is most likely one of seed predation. Although it is very possible that the seeds and pulp of the plant are cytotoxic (they showed the highest death rate of any of the extracts), a lack of secondary chemistry could perhaps be due to the physical protection its hard shell provides. It is interesting to note that the two plants which showed anti-bacterial activity were the two in which the seeds were too small to be separated from the pulp of the fruit and were eaten whole by the monkeys. The bioactivity may be simply a defense mechanism against bacteria, or it may suggest the presence of compounds beneficial to the monkeys to encourage dispersal. The secondary compounds of P. alata, on the other hand, are most likely to be beneficial through their anti-oxidant properties and other medicinal properties similar to those of guarana (P. cupana).

The hypothesis set forth by this study is that S. sciureus selects for food plants based on beneficial secondary chemistry as well as their energy/nutrition content. Earlier experiments have shown that generally, S. sciureus is an opportunistic feeder, whose goal is to maximize carbohydrate content (Laska 2001). But these experiments do not take into account the possibility of monkeys actively seeking secondary compounds. One of the few documented cases of squirrel monkeys actively seeking secondary compounds involved the observation of S. sciureus self-administration of plants that contain THC (Tanda et al. 2000). So although zoopharmacognostic behavior has yet to be observed in wild S. sciureus, it remains a possibility-and a rather likely one considering the vast array of plants to select from in the flooded forests of the Amazon, each with a vast array of secondary compounds. The pressures to develop this type of behavior are extreme in the difficult life conditions of the Amazon. A relationship in which monkeys disperse seeds in order to receive secondary medicinal benefits as well as nutritional benefits is plausible, but more study must be conducted to test this hypothesis.

References

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Belliardo, F, et al, 1985. HPLC determination of caffeine and theophylline in Paullinia cupana Kunth (guarana) and Cola spp. samples. Z Lebensm Unters Forsch, 180(5):398-401.

Bydlowsky, SP, et al, 1988. A novel property of an aqueous guarana extract (Paullinia cupana): inhibition of platelet aggregation in vitro and in vivo. Brazilian Journal of Medical Biological Research, 21(3):535-8.

Espinola, EB, et al, 1997. Pharmacological activity of Guarana (Paullinia cupana Mart.) in laboratory animals. Journal of Ethnopharmacology. 55(3):223-9.

Laska, M. 2001. A comparison of food preferences and nutrient composition in captive squirrel monkeys,

Mattei, R, et al, 1998. Guarana (Paullinia cupana): toxic behavioral effects in laboratory animals and antioxidants activity in vitro. Journal of Ethnopharmacology, 60(2):111-6.

Perez-Guerrero, C, et al, 2001. A pharmacological study of Cecropia obtusifolia Bertol aqueous extract. Journal of Ethnopharmacology, 76(3):279-84.

Saimiri sciureus, and pigtail macaques, Macaca nemestrina. Physiology and Behavior, 73(1-2):111-20.

Salvat, A, et al. 2001. Screening of some plants from Northern Argentina for their antimicrobial activity. Letters in Applied Microbiology, 32(5):293-7.

Santa Maria, A, et al, 1998. Evaluation of the toxicity of guarana with in vitro bioassays. Ecotoxicology and Environmental Safety. 39(3):164-7.

Tanda, G, et al. 2000. Self-administration behavior is maintained by the psychoactive ingredient of marijuana in squirrel monkeys. Nature Neuroscience, 3(11):1073-4.