ANTI-FUNGAL PLANTS OF THE PERUVIAN
AMAZON: A SURVEY OF ETHNOMEDICAL USES AND BIOLOGICAL ACTIVITY
A. HERFORTH, J. RUIZ, E. MOSQUERA, M. LAUX, E. RODRIGUEZ
A. HERFORTH, J. RUIZ, E. MOSQUERA, M. LAUX, E. RODRIGUEZ
Fungal infections are a constant problem for people living
in the rainforest, due to the climate’s intense heat and moisture. These
ideal growing conditions for fungi present a challenge for plants as well,
which use chemical defenses to avoid fungal diseases. The anti-fungal
secondary metabolites produced not only protect the plants, but can also
help people cure mycoses. The botanical treatments of skin mycoses and
vaginal yeast infections used among villagers of Jaldar and Jerusalen
on the Yarapa River, Loreto, Peru are presented in this study. In addition,
a preliminary analysis of the chemistry and bioactivity of the anti-fungal
plants was conducted. Treatments of skin mycoses included in this study
are preparations of Vismia angusta (Clusiaceae), Senna reticulata (Fabaceae),
Senna macrophylla (Fabaceae), Genipa americana (Rubiaceae), and Cecropia
membranacea (Cecropiaceae) and the beetle larvae that eat it. Remedies
for vaginal yeast infections, used separately and in mixtures by midwives
are Spondias mombin (Anacardiaceae), Maytenus macrocarpa (Celastraceae),
Campsiandra spruceana (Fabaceae), Psidium guajava (Myrtaceae), Coussapoa
nitida (Cecropiaceae), Brosimum acutifolium (Moraceae), Brosimum rubescens
(Moraceae), Swartzia polyphylla (Fabaceae), Anacardium giganteum (Anacardiaceae),
Maquira coreacea (Moraceae), Croton lechleri (Euphorbiaceae), Copaifera
paupera (Fabaceae), Chenopodium ambrosioides (Chenopodiaceae), and an
unidentified white fungus that grows on dead, dry tree branches. Disk
diffusion assays were conducted against two yeasts, Candida albicans and
Saccharomyces cerevisiae, three dermatophytes, Epidermophyton floccosum,
Trichophyton mentagrophytes, and Microsporum canis, two gram-positive
bacteria, Bacillus cereus and Staphylococcus aureus, and three gram-negative
bacteria, Pseudomonas aeruginosa, Escherichia coli, and H. pylori. All
extracts tested except M. coreacea and C. ambrosioides showed anti-fungal
activity against C. albicans, the fungus responsible for yeast infections.
Some of the plants were active against the dermatophytes, the most active
being a mix for yeast infections made a midwife and C. paupera. Many of
the extracts were active in the bioassays against the other microbes as
well, C. paupera being the most uniformly anti-bacterial (active against
all bacteria). In addition, a crystal violet bioassay was conducted on
skin and colon cancer cell lines to assess the cytotoxicity associated
with the use of these anti-fungal plants. On the whole, the plants were
not found to be cytotoxic. The only two significantly toxic plants, C.
paupera and C. lechleri, are heavily diluted when used medicinally.
The Yarapa River is a small tributary to the Amazon, unmarked on most maps, between Nauta and Iquitos in the state of Loreto, Peru. For five weeks in the summer of 2001, the people of two villages on the river (Jaldar and Jerusalen) worked with us to compile information about the plants they use to treat illness. Knowledge of medicinal plants was passed down from family members or elders to informants when they had shown a personal interest in and an aptitude for healing. The main informants for this study were a forest guide (Estéban Mosquera), a botanist (Juan Ruiz), three midwives from the villages on site (Felicita, Leocha, and Jorge), and two merchants in the medicinal plants marketplace in Iquitos. Through conversations and interviews, a list of anti-fungal treatments was generated and plants were collected for study.
This study focuses on treatments for mycoses, especially those used to treat ringworm caused by Epidermophyton floccosum, Microsporum canis and Trichophyton mentagrophytes, and yeast infections caused by Candida albicans. In this study, medicinal plants identified by informants were screened for bioactivity against these and other microbes. Many medicinal plants are used for multiple purposes due to their strong bioactivity. Plants that were (a) used against ulcers or stomachache in addition to their anti-fungal use or (b) ingested treatments that were active against gram-negative bacteria were screened for activity against Helicobacter pylori, a gram-negative bacteria that causes ulcers. This wide-scale survey will provide insight into the botanical medicines used in the Amazon.
ETHNOBOTANY
Formal interviews with the midwives were conducted on three occasions, and notes were recorded. Informal discussion with our guide, Estéban Mosquera, botanist Juan Ruiz, and chemist Julio Arce (University of Iquitos) took place over the five-week study period. The following is a description of the information gathered during the interviews and discussions; it is summarized in Table 1.
Felicita has been a midwife for many years, and learned the use of medicinal plants from her mother. She shared the two treatments she prescribes to treat yeast infections and one treatment for ringworm. In one treatment against yeast infections, she boils together the chopped bark of Maytenus macrocarpa, Campsiandra spruceana, Spondias mombin, Brosimum acutifolium, Psidium guajava, and Anacardium sp. in equal parts in about a liter of water. The first three plants are the most essential, and the rest are added more casually. Croton lechleri is another good addition, but it only grows in the high forests (not the varsia forests of Jaldar), so it is not often available. A patient suffering from a yeast infection will drink the mixture diluted in water or aguardiente in the morning, and wash with it in the afternoon for eight days, or until the infection stops. Another treatment she uses is a little cup of bitter resin from Maquira coreacea, pure or in aguardiente, three times a day until the patient is cured. Cecropia membranacea and the unidentified beetle larvae (“gusanitos”) that eat them are good for ringworm. According to Felicita, the gusanitos are better because they concentrate the medicine. She collected the beetle larvae for the experiment.
Leocha is younger than Felicita, but has as much experience, having twenty of her own children. She provided information for treatment of yeast infections. She prescribes “florblanco,” a white fungus that grows on dry branches, to be used as a wash two times a day, before and after sleep because infections are worst at night. Another reliable plant is S. mombin, boiled with or without M. macrocarpa and guamas (“water lettuce”), and drunk or used as a wash two times a day to control itching and cure the yeast infection.
Jorge, the only male “midwife” or nurse, was serious about his work and described exact recipes for treatments of yeast infections. He mixes the barks of M. macrocarpa, S. mombin, Coussapoa nitida, and Anacardium giganteum, and scrapings of the inner wood of Brosimum rubescens and Swartzia polyphylla. A. giganteum and B. rubescens are not locally available in the varsia forest, but Jorge keeps stocks of them to use as he needs them. These ingredients are all mashed and boiled together for about five minutes, or until the water level drops by two finger-widths. The mixture is cooled, poured into a bottle and given to the patient with instructions to drink it three times a day and to wash with it twice a day. The treatment should not be used for more than 15 days, and the infection should clear by then. The treatment should be used two times more after the infection stops.
The guides (Estéban Mosquera, Juan Ruiz) provided significant information about anti-fungal plants, especially about those used for dermal mycoses. The bark of Senna reticulata can be boiled for an anti-fungal treatment. The leaves and bark contain emodins (anthraquinones), the odor of which is said to kill chicken lice (Arce, personal interview). Senna macrophylla has an anti-fungal yellow resin from its fruit pod that cures mycoses and acts as a repellent when applied directly to skin. Genipa americana has a fruit that can be rubbed onto the skin and oxidized into a black dye that is both a fungicide and a mosquito repellent; the fruit can also be eaten to cure bronchitis. The sticky yellow latex of Vismia angusta contains hypericins (Arce, personal interview). Esteban Mosquera mixes the resin with Vaseline for use as an anti-fungal cream. Cecropia membranacea leaves contain an anti-fungal hormone, ponasterone, which also has anti-inflammatory properties (Arce, personal interview). Beetle larvae eat it and are a more powerful anti-fungal treatment.
As mentioned above, some plants were bought at the medicinal plant marketplace in Iquitos, because they were not available at the field site. Two women sold the plants used in this study, and talked about their properties and uses. Many plants are sold fresh, and many are sold as extractions. The bark for sale can be heated in water to make a tea, or “extracted” in aguardiente for 7-10 days, and both are equal treatments. (The validates the extraction methods of this study, since many treatments that were supposed to be boiled were extracted in ethanol.) Croton lechleri is used against fungal infections, gastritis, throat infections, and other maladies. It is useful as a vaginal wash twice a day for yeast infections or uterus infections, but it was said that S. mombin is a better cure. Used as a drink, 3-5 drops of either of the concentrated liquids are mixed with water, and drunk twice a day. Copaifera paupera is used for yeast infections, as well as dermal mycoses, cuts, ulcers, and many other afflictions. It is a pungent, yellow resin that is diluted in water when taken internally or used as a wash. Chenopodium ambrosioides also prevents fungal infections. The leaves of this herb are boiled in two cups of water with a little salt, and the infusion is then used to wash infected wounds; it is also good to treat headaches. One of the women at the market, Norma, made a special mixture that would cure yeast infections when drunk three times a day, before meals. Her mixture consisted of S. mombin, a plant called “renaquilla” (probably C. nitida), Psittacanthus sp., and Piper arboreum.
MATERIALS AND METHODS
Preliminary trials (Yarapa and Punta Cana)
Plants were identified and gathered with the help of botanist Juan Ruiz. Fresh material was macerated and extracted in 96% ethanol for 1-2 days, filtered through a cotton plug, and concentrated using a hairdryer. Most plants were ground and placed in ethanol alone. However, due to the possibility of synergistic effects of the combination of plants in the midwives’ mixtures, two mixed extracts were prepared according to the recipes that Felicita and Jorge use for patients suffering from yeast infections. For each mixture, equal amounts of each plant were combined and extracted together. Jorge also made and contributed his own preparation of the mixture he prescribes. Felicita collected the unidentified beetle larvae used against ringworm and Leocha collected the unidentified white fungus used against yeast infections. Because certain plants were unavailable, several extracts were obtained at the medicinal plant marketplace in Iquitos, the regional capital. These include Brosimum rubescens, Anacardium giganteum, Croton lechleri, Copaifera paupera, and a mix sold by a merchant specifically for yeast infections, including Spondias mombin, Psittacanthus sucuuba, Coussapoa nitida, and Piper arboreum (Piperaceae). Chenopodium ambrosioides was obtained fresh at the marketplace.
Extracts were sent to the Cornell-Punta Cana Biodiversity Laboratory in Punta Cana, Dominican Republic for bioactivity screening. Anti-microbial disk diffusion assays were performed on the crude extracts at unknown concentrations. Microbes used in the assays were the yeast Candida albicans, the gram-positive bacteria Bacillus cereus, and the gram-negative bacteria Pseudomonas aeruginosa. Due to delays in availability, not all extracts were preliminarily tested for anti-microbial activity.
Continued research (Cornell University)
The extracts made in Peru were brought to Cornell University, where they were further studied. The crude extracts were filtered using filter paper and a büchner funnel under vacuum pressure. They were then dried using a rotary evaporator, and redissolved in 70% ethanol at a standard concentration of 1mL solvent/25mg dry weight. In the cases of Psidium guajava, Coussapoa nitida, the beetle larvae, and “Norma’s mix,” there was very little dry material, so extracts were redissolved in 1mL solvent/10mg dry weight so that there would be enough material to complete the tests.
Microbes used in the disk diffusion assays were the yeasts Candida albicans (ATCC#90028) grown on Sabouraud dextrose agar (Difco 0109) and Saccharomyces cerevisiae (ATCC#2601) grown on YM agar (Difco 0712); three mycelial fungi, Epidermophyton floccosum, Trichophyton mentagrophytes, and Microsporum canis, all grown on Sabouraud dextrose agar; two gram-positive bacteria, Bacillus cereus (ATCC#11778) grown on nutrient agar (Difco 0001) and Staphylococcus aureus (ATCC#25923) grown on trypticase soy agar (BBL 11043); and three gram-negative bacteria, Pseudomonas aeruginosa (ATCC#27853) grown on nutrient agar (Difco 0001), Escherichia coli (ATCC#25922) grown on Bacto-Tryptone 10g/1L, Bacto-Yeast Ext .5g/1L, NaCl 10g/1L, Bactoagar 15g/1L, and Helicobacter pylori, grown on nutrient agar (Difco 0001) ??
The prepared media was autoclaved, poured onto petri dishes and allowed to solidify. Plates of nutrient agar were inoculated with microbial culture. Sterile disks were impregnated with ???L of plant extract and allowed to dry. The disks were then placed on the inoculated plates, along with blank negative control disks. Plates of all microbes except the mycelial fungi were incubated for 24 hours at 37?C (body temperature) and zones of inhibition were observed. Assays were repeated to ensure accuracy. The mycelial fungi T. mentagrophytes and E. floccosum were incubated for 48 hours at 35?C. M. canis was incubated for 72 hours at 35?C.
Cancer cell lines HT29 (human colon cancer) and A431 (human epithelial skin cancer) were tested for induction of cytotoxicity (necrosis) by a range of concentrations of the extracts. Cell necrosis caused by addition of extracts was assessed using the crystal violet bioassay, as described by Laux (2001). Extracts were dried and redissolved in di-methyl sulfoxide (DMSO) for this assay. Extracts tested included the externally applied V. angusta, S. reticulata, G. americana, C. membranacea. The effect of these plants on skin cancer line A431 was of particular interest, since they are applied to the skin. Extracts tested that were applied both externally as a wash and imbibed included Felicita’s mix and its components individually (the barks of P. guajava, C. spruceana, B. acutifolium, M. macrocarpa, and S. mombin); Jorge’s mix (made by Jorge); S. polyphylla; B. rubescens; C. lechleri; and C. paupera. The effect of these plants on both the skin cancer and colon cancer lines was of interest, since they are both applied to the skin and pass through the gut. Blank and DMSO controls were tested.
RESULTS AND DISCUSSION
The rings of inhibition on the plates were observed in the disk diffusion assays, and results of both the preliminary and standardized trials are summarized in Table 2 (Anti-fungal activity) and Table 3 (Anti-bacterial activity). It should be noted that disk diffusion assays are qualitative, not quantitative, due to the fact that inhibition depends on not only the concentration of each extract, but also the ability of each extract to be absorbed into the media.
Preliminary and continued results sometimes differed. This can best be explained by variations in extract concentration between the preliminary trials and the standardized trials. Although the concentrations for the preliminary trials were unknown, based on the thickness of the extracts, most were probably more concentrated for the preliminary trials.
All extracts except for C. ambrosioides, M. coreacea, S. macrophylla, and the unidentified beetle larvae inhibited the growth of C. albicans at standardized concentration. All except these in addition to C. membranacea and G. americana inhibited S. cerevisiae.
The plants used as treatments for dermal mycoses that were active against at least one of the species tested that commonly causes mycoses (Microsporum canis, Epidermohpyton floccosum and Trichophyton mentagrophytes) are C. membranacea, C. lechleri, and G. americana. Several of the anti-yeast infection treatments also proved to be active against the dermatophytes, including B. acutifolium, C. paupera, M. coreacea, Jorge’s mix (both the one made by Jorge and the one made to imitate it), Norma’s mix, P. guajava (both bark and leaves), and S. mombin. The M. canis assay was difficult to interpret due to thick, uneven growth. It should be repeated under different kinds of conditions (incubation temperature, amount of fungus inoculated, humidity) to determine a protocol that is successful for disk diffusion assays on M. canis.
The extracts were generally quite inactive against bacteria, although some were very active against P. aeruginosa. This may further confirm the plants’ activity against fungal infections, since normally occurring bacteria on the human body often impede the proliferation of pathogenic fungi (Todar 2002). If the extracts inhibited the growth of these “good” bacteria, their efficacy against fungi would be compromised.
Extracts that were active against the ulcer-causing bacteria H. pylori were A. giganteum, B. acutifolium, C. spruceana, C. paupera, C. lechleri, Felicita’s mix, Jorge’s mix, M. macrocarpa, P. guajava leaves, and S. polyphylla. The following plants were not found to be active: B. rubescens, C. mebranacea, C. ambrosioides, C. nitida, G. americana, Norma’s mix, P. guajava bark, and S. mombin.
In the crystal violet assays to test for cancer cell necrosis (cytotoxicity), the extracts were generally non-toxic. The initial concentration of extract contacting the cells was 9.1-3?g/?L solution + media. The point of recovery of the both cell lines for almost all extract treatments was the second or third dilution in a serial dilution. Significantly cytotoxic extracts were C. lechleri, which killed HT29 cells until the sixth dilution and A431 cells until the fourth dilution, and C. paupera, which killed all cells in both lines through 16 dilutions. It is useful to note that these two most cytotoxic plants are known as powerful plants in the Yarapa region and are used sparingly. According to the marketplace vendors, if they are consumed, only two drops are mixed with water or aguardiente (sugar cane rum). When C. lechleri is used as a vaginal wash, it is diluted in plenty of warm water. These were the only two plants in this study for which extraordinary caution was urged.
The cytotoxicity of both C. lechleri and C. paupera indicate promise of anti-cancer activity. The toxicity of C. lechleri, especially to the colon cancer cells, compliments its activity against H. pylori, a bacterium that is linked to stomach and intestinal cancers.
This study scientifically shows that almost all of the treatments for fungal infections used by the people of the Yarapa region do indeed inhibit fungal growth.
WORKS CITED
Arce, Julio, phytochemist at the University of Iquitos. Personal interviews. 2001.
Laux, M. 2001. The Induction of Apoptosis by Selected Natural Products in Human Breast Cancer in Vitro. Ch. II. PhD dissertation in review.
Todar, Kenneth. 2002. “The Normal Bacterial Flora of Animals.” University of Wisconsin Department of Bacteriology. Accessed online, April 2002 at http://www.bact.wisc.edu/Bact330/lecturenf.
The Yarapa River is a small tributary to the Amazon, unmarked on most maps, between Nauta and Iquitos in the state of Loreto, Peru. For five weeks in the summer of 2001, the people of two villages on the river (Jaldar and Jerusalen) worked with us to compile information about the plants they use to treat illness. Knowledge of medicinal plants was passed down from family members or elders to informants when they had shown a personal interest in and an aptitude for healing. The main informants for this study were a forest guide (Estéban Mosquera), a botanist (Juan Ruiz), three midwives from the villages on site (Felicita, Leocha, and Jorge), and two merchants in the medicinal plants marketplace in Iquitos. Through conversations and interviews, a list of anti-fungal treatments was generated and plants were collected for study.
This study focuses on treatments for mycoses, especially those used to treat ringworm caused by Epidermophyton floccosum, Microsporum canis and Trichophyton mentagrophytes, and yeast infections caused by Candida albicans. In this study, medicinal plants identified by informants were screened for bioactivity against these and other microbes. Many medicinal plants are used for multiple purposes due to their strong bioactivity. Plants that were (a) used against ulcers or stomachache in addition to their anti-fungal use or (b) ingested treatments that were active against gram-negative bacteria were screened for activity against Helicobacter pylori, a gram-negative bacteria that causes ulcers. This wide-scale survey will provide insight into the botanical medicines used in the Amazon.
ETHNOBOTANY
Formal interviews with the midwives were conducted on three occasions, and notes were recorded. Informal discussion with our guide, Estéban Mosquera, botanist Juan Ruiz, and chemist Julio Arce (University of Iquitos) took place over the five-week study period. The following is a description of the information gathered during the interviews and discussions; it is summarized in Table 1.
Felicita has been a midwife for many years, and learned the use of medicinal plants from her mother. She shared the two treatments she prescribes to treat yeast infections and one treatment for ringworm. In one treatment against yeast infections, she boils together the chopped bark of Maytenus macrocarpa, Campsiandra spruceana, Spondias mombin, Brosimum acutifolium, Psidium guajava, and Anacardium sp. in equal parts in about a liter of water. The first three plants are the most essential, and the rest are added more casually. Croton lechleri is another good addition, but it only grows in the high forests (not the varsia forests of Jaldar), so it is not often available. A patient suffering from a yeast infection will drink the mixture diluted in water or aguardiente in the morning, and wash with it in the afternoon for eight days, or until the infection stops. Another treatment she uses is a little cup of bitter resin from Maquira coreacea, pure or in aguardiente, three times a day until the patient is cured. Cecropia membranacea and the unidentified beetle larvae (“gusanitos”) that eat them are good for ringworm. According to Felicita, the gusanitos are better because they concentrate the medicine. She collected the beetle larvae for the experiment.
Leocha is younger than Felicita, but has as much experience, having twenty of her own children. She provided information for treatment of yeast infections. She prescribes “florblanco,” a white fungus that grows on dry branches, to be used as a wash two times a day, before and after sleep because infections are worst at night. Another reliable plant is S. mombin, boiled with or without M. macrocarpa and guamas (“water lettuce”), and drunk or used as a wash two times a day to control itching and cure the yeast infection.
Jorge, the only male “midwife” or nurse, was serious about his work and described exact recipes for treatments of yeast infections. He mixes the barks of M. macrocarpa, S. mombin, Coussapoa nitida, and Anacardium giganteum, and scrapings of the inner wood of Brosimum rubescens and Swartzia polyphylla. A. giganteum and B. rubescens are not locally available in the varsia forest, but Jorge keeps stocks of them to use as he needs them. These ingredients are all mashed and boiled together for about five minutes, or until the water level drops by two finger-widths. The mixture is cooled, poured into a bottle and given to the patient with instructions to drink it three times a day and to wash with it twice a day. The treatment should not be used for more than 15 days, and the infection should clear by then. The treatment should be used two times more after the infection stops.
The guides (Estéban Mosquera, Juan Ruiz) provided significant information about anti-fungal plants, especially about those used for dermal mycoses. The bark of Senna reticulata can be boiled for an anti-fungal treatment. The leaves and bark contain emodins (anthraquinones), the odor of which is said to kill chicken lice (Arce, personal interview). Senna macrophylla has an anti-fungal yellow resin from its fruit pod that cures mycoses and acts as a repellent when applied directly to skin. Genipa americana has a fruit that can be rubbed onto the skin and oxidized into a black dye that is both a fungicide and a mosquito repellent; the fruit can also be eaten to cure bronchitis. The sticky yellow latex of Vismia angusta contains hypericins (Arce, personal interview). Esteban Mosquera mixes the resin with Vaseline for use as an anti-fungal cream. Cecropia membranacea leaves contain an anti-fungal hormone, ponasterone, which also has anti-inflammatory properties (Arce, personal interview). Beetle larvae eat it and are a more powerful anti-fungal treatment.
As mentioned above, some plants were bought at the medicinal plant marketplace in Iquitos, because they were not available at the field site. Two women sold the plants used in this study, and talked about their properties and uses. Many plants are sold fresh, and many are sold as extractions. The bark for sale can be heated in water to make a tea, or “extracted” in aguardiente for 7-10 days, and both are equal treatments. (The validates the extraction methods of this study, since many treatments that were supposed to be boiled were extracted in ethanol.) Croton lechleri is used against fungal infections, gastritis, throat infections, and other maladies. It is useful as a vaginal wash twice a day for yeast infections or uterus infections, but it was said that S. mombin is a better cure. Used as a drink, 3-5 drops of either of the concentrated liquids are mixed with water, and drunk twice a day. Copaifera paupera is used for yeast infections, as well as dermal mycoses, cuts, ulcers, and many other afflictions. It is a pungent, yellow resin that is diluted in water when taken internally or used as a wash. Chenopodium ambrosioides also prevents fungal infections. The leaves of this herb are boiled in two cups of water with a little salt, and the infusion is then used to wash infected wounds; it is also good to treat headaches. One of the women at the market, Norma, made a special mixture that would cure yeast infections when drunk three times a day, before meals. Her mixture consisted of S. mombin, a plant called “renaquilla” (probably C. nitida), Psittacanthus sp., and Piper arboreum.
MATERIALS AND METHODS
Preliminary trials (Yarapa and Punta Cana)
Plants were identified and gathered with the help of botanist Juan Ruiz. Fresh material was macerated and extracted in 96% ethanol for 1-2 days, filtered through a cotton plug, and concentrated using a hairdryer. Most plants were ground and placed in ethanol alone. However, due to the possibility of synergistic effects of the combination of plants in the midwives’ mixtures, two mixed extracts were prepared according to the recipes that Felicita and Jorge use for patients suffering from yeast infections. For each mixture, equal amounts of each plant were combined and extracted together. Jorge also made and contributed his own preparation of the mixture he prescribes. Felicita collected the unidentified beetle larvae used against ringworm and Leocha collected the unidentified white fungus used against yeast infections. Because certain plants were unavailable, several extracts were obtained at the medicinal plant marketplace in Iquitos, the regional capital. These include Brosimum rubescens, Anacardium giganteum, Croton lechleri, Copaifera paupera, and a mix sold by a merchant specifically for yeast infections, including Spondias mombin, Psittacanthus sucuuba, Coussapoa nitida, and Piper arboreum (Piperaceae). Chenopodium ambrosioides was obtained fresh at the marketplace.
Extracts were sent to the Cornell-Punta Cana Biodiversity Laboratory in Punta Cana, Dominican Republic for bioactivity screening. Anti-microbial disk diffusion assays were performed on the crude extracts at unknown concentrations. Microbes used in the assays were the yeast Candida albicans, the gram-positive bacteria Bacillus cereus, and the gram-negative bacteria Pseudomonas aeruginosa. Due to delays in availability, not all extracts were preliminarily tested for anti-microbial activity.
Continued research (Cornell University)
The extracts made in Peru were brought to Cornell University, where they were further studied. The crude extracts were filtered using filter paper and a büchner funnel under vacuum pressure. They were then dried using a rotary evaporator, and redissolved in 70% ethanol at a standard concentration of 1mL solvent/25mg dry weight. In the cases of Psidium guajava, Coussapoa nitida, the beetle larvae, and “Norma’s mix,” there was very little dry material, so extracts were redissolved in 1mL solvent/10mg dry weight so that there would be enough material to complete the tests.
Microbes used in the disk diffusion assays were the yeasts Candida albicans (ATCC#90028) grown on Sabouraud dextrose agar (Difco 0109) and Saccharomyces cerevisiae (ATCC#2601) grown on YM agar (Difco 0712); three mycelial fungi, Epidermophyton floccosum, Trichophyton mentagrophytes, and Microsporum canis, all grown on Sabouraud dextrose agar; two gram-positive bacteria, Bacillus cereus (ATCC#11778) grown on nutrient agar (Difco 0001) and Staphylococcus aureus (ATCC#25923) grown on trypticase soy agar (BBL 11043); and three gram-negative bacteria, Pseudomonas aeruginosa (ATCC#27853) grown on nutrient agar (Difco 0001), Escherichia coli (ATCC#25922) grown on Bacto-Tryptone 10g/1L, Bacto-Yeast Ext .5g/1L, NaCl 10g/1L, Bactoagar 15g/1L, and Helicobacter pylori, grown on nutrient agar (Difco 0001) ??
The prepared media was autoclaved, poured onto petri dishes and allowed to solidify. Plates of nutrient agar were inoculated with microbial culture. Sterile disks were impregnated with ???L of plant extract and allowed to dry. The disks were then placed on the inoculated plates, along with blank negative control disks. Plates of all microbes except the mycelial fungi were incubated for 24 hours at 37?C (body temperature) and zones of inhibition were observed. Assays were repeated to ensure accuracy. The mycelial fungi T. mentagrophytes and E. floccosum were incubated for 48 hours at 35?C. M. canis was incubated for 72 hours at 35?C.
Cancer cell lines HT29 (human colon cancer) and A431 (human epithelial skin cancer) were tested for induction of cytotoxicity (necrosis) by a range of concentrations of the extracts. Cell necrosis caused by addition of extracts was assessed using the crystal violet bioassay, as described by Laux (2001). Extracts were dried and redissolved in di-methyl sulfoxide (DMSO) for this assay. Extracts tested included the externally applied V. angusta, S. reticulata, G. americana, C. membranacea. The effect of these plants on skin cancer line A431 was of particular interest, since they are applied to the skin. Extracts tested that were applied both externally as a wash and imbibed included Felicita’s mix and its components individually (the barks of P. guajava, C. spruceana, B. acutifolium, M. macrocarpa, and S. mombin); Jorge’s mix (made by Jorge); S. polyphylla; B. rubescens; C. lechleri; and C. paupera. The effect of these plants on both the skin cancer and colon cancer lines was of interest, since they are both applied to the skin and pass through the gut. Blank and DMSO controls were tested.
RESULTS AND DISCUSSION
The rings of inhibition on the plates were observed in the disk diffusion assays, and results of both the preliminary and standardized trials are summarized in Table 2 (Anti-fungal activity) and Table 3 (Anti-bacterial activity). It should be noted that disk diffusion assays are qualitative, not quantitative, due to the fact that inhibition depends on not only the concentration of each extract, but also the ability of each extract to be absorbed into the media.
Preliminary and continued results sometimes differed. This can best be explained by variations in extract concentration between the preliminary trials and the standardized trials. Although the concentrations for the preliminary trials were unknown, based on the thickness of the extracts, most were probably more concentrated for the preliminary trials.
All extracts except for C. ambrosioides, M. coreacea, S. macrophylla, and the unidentified beetle larvae inhibited the growth of C. albicans at standardized concentration. All except these in addition to C. membranacea and G. americana inhibited S. cerevisiae.
The plants used as treatments for dermal mycoses that were active against at least one of the species tested that commonly causes mycoses (Microsporum canis, Epidermohpyton floccosum and Trichophyton mentagrophytes) are C. membranacea, C. lechleri, and G. americana. Several of the anti-yeast infection treatments also proved to be active against the dermatophytes, including B. acutifolium, C. paupera, M. coreacea, Jorge’s mix (both the one made by Jorge and the one made to imitate it), Norma’s mix, P. guajava (both bark and leaves), and S. mombin. The M. canis assay was difficult to interpret due to thick, uneven growth. It should be repeated under different kinds of conditions (incubation temperature, amount of fungus inoculated, humidity) to determine a protocol that is successful for disk diffusion assays on M. canis.
The extracts were generally quite inactive against bacteria, although some were very active against P. aeruginosa. This may further confirm the plants’ activity against fungal infections, since normally occurring bacteria on the human body often impede the proliferation of pathogenic fungi (Todar 2002). If the extracts inhibited the growth of these “good” bacteria, their efficacy against fungi would be compromised.
Extracts that were active against the ulcer-causing bacteria H. pylori were A. giganteum, B. acutifolium, C. spruceana, C. paupera, C. lechleri, Felicita’s mix, Jorge’s mix, M. macrocarpa, P. guajava leaves, and S. polyphylla. The following plants were not found to be active: B. rubescens, C. mebranacea, C. ambrosioides, C. nitida, G. americana, Norma’s mix, P. guajava bark, and S. mombin.
In the crystal violet assays to test for cancer cell necrosis (cytotoxicity), the extracts were generally non-toxic. The initial concentration of extract contacting the cells was 9.1-3?g/?L solution + media. The point of recovery of the both cell lines for almost all extract treatments was the second or third dilution in a serial dilution. Significantly cytotoxic extracts were C. lechleri, which killed HT29 cells until the sixth dilution and A431 cells until the fourth dilution, and C. paupera, which killed all cells in both lines through 16 dilutions. It is useful to note that these two most cytotoxic plants are known as powerful plants in the Yarapa region and are used sparingly. According to the marketplace vendors, if they are consumed, only two drops are mixed with water or aguardiente (sugar cane rum). When C. lechleri is used as a vaginal wash, it is diluted in plenty of warm water. These were the only two plants in this study for which extraordinary caution was urged.
The cytotoxicity of both C. lechleri and C. paupera indicate promise of anti-cancer activity. The toxicity of C. lechleri, especially to the colon cancer cells, compliments its activity against H. pylori, a bacterium that is linked to stomach and intestinal cancers.
This study scientifically shows that almost all of the treatments for fungal infections used by the people of the Yarapa region do indeed inhibit fungal growth.
WORKS CITED
Arce, Julio, phytochemist at the University of Iquitos. Personal interviews. 2001.
Laux, M. 2001. The Induction of Apoptosis by Selected Natural Products in Human Breast Cancer in Vitro. Ch. II. PhD dissertation in review.
Todar, Kenneth. 2002. “The Normal Bacterial Flora of Animals.” University of Wisconsin Department of Bacteriology. Accessed online, April 2002 at http://www.bact.wisc.edu/Bact330/lecturenf.