ETHNOBOTANY AND BIOLOGICAL ACTIVITY
OF PLANTS UTILIZED DURING PREGNANCY AND CHILDBIRTH IN THE PERUVIAN AMAZON
A. KRAMER, E. MOSQUERA, J. RUIZ, E. RODRIGUEZ
A. KRAMER, E. MOSQUERA, J. RUIZ, E. RODRIGUEZ
ABSTRACT
Peruvian women of the Amazon region have an average of 7.9 children, more than double the national average of 3.4 children. For these women, traditional methods of childbirth continue to be the primary means of healthcare. The collection and use of plants in the Peruvian Amazon by midwives is part of this tradition that has been sustained for centuries. The objective of this study is to evaluate natural products used during pregnancy and childbirth for their medicinal value. Structured interviews were conducted with three midwives in the communities of Jaldar and Jerusalen along the Yarapa River in the Loreto region of Peru. Ethnobotanical data was collected from the midwives and categorized into four treated conditions: maintenance of wellness during pregnancy, induction of labor or contractions, pain during childbirth, and postpartum infections of both mother and child. Ten plants common to each interview were identified and studied: Spondias mombin (Anacardiaceae), Pouteria caimito (Sapotaceae), Gossipium barbadense (Malvaceae), Gomphaena globosa (Amaranthaceae), Zingiber officinalis (Zingiberaceae), Malacra rudelara (Malvaceae), Piper peltatum (Piperaceae), Cymbogon citratus (Poaceae), Cestrum hediondinim (Solanaceae), and Bixa orellana (Bixaceae). Plants were extracted with 96% ethanol and chemical analysis was conducted with thin-layer chromatography (TLC). Preliminary results report anti-oxidant activity in Spondias mombin, Pouteria caimito, Gossipium barbadense, Malacra rudelara, Piper peltatum, Cestrum hediondinim and Bixa orellana. A brine shrimp assay indicated high levels of cytotoxicity in Bixa orellana, Malachra rudelara, and Piper peltatum. Biological activity was assessed using anti-bacterial and anti-fungal assays. Bacterial bioassays on Pseudomonas aeruginosa (gram-negative) and Bacillus cereus (gram-positive) revealed moderate growth inhibition by Bixa orellana, Pouteria caimito, Spondias mombin, and Gossipium barbadense. These four extracts also showed moderate growth inhibition of the yeast Candida albicans. High inhibition of Candida albicans was effected by both Piper peltatum and Zingiber officinalis.
There are two confounding factors that contribute to the high reproductive rate in the Amazon region of Peru. The first is the high number of children born to each woman in this region. These women have an average of 7.9 children, more than double the national average of 3.4 children (Compendium 1992). Particularly, in the Achual community of Jersalen where we studied, an average of 8.0 children are born to every woman (Compendium 1992). The second factor and one of the highest risks of reproduction in indigenous women is early childbearing. Among Amazonian girls ages 12-14, 2.3% are already mothers, a number nearly twice the national average. In adolescents ages 15-19, the number of mothers jumps to 34.4%, more than triple the percentage seen on a national level (Comportamiento 1996).
These statistics suggest the large role a midwife must perform in the community. Their work involves the collection and careful selection of herbal medicines used during pregnancy and childbirth. They determine the preparation in administering remedies to women in labor. Perhaps the most important role they perform is the passing on of knowledge of traditional medicines to others in their community (Sáenz 2000).
The objective of this study is to evaluate the natural products used by midwives and access their biological activity.
MATERIALS AND METHODS
Ethnobotanical data was collected through structured interviews with midwives in the communities of Jaldar and Jerusalen. Interviews were conducted with two midwives in Jaldar and one midwife in Jerusalen. Initially, each midwife was asked to describe specific plants used during pregnancy or childbirth. Further clarifications were made regarding dosage and preparation. In addition, voucher specimens were taken of each of the plants. A second follow-up interview was conducted with each midwife to specify exact uses and dosages.
Fresh green material was collected for each species studied. Leaves were ground and then extracted in 97% EtOH for 24 hours. Extracts were then filtered and dried using hot air to concentrate the samples.
A preliminary chemical analysis was performed using thin-layer chromatography (TLC). Extracts were spotted on aluminum-backed TLC plates coated with silica gel. Three solvent systems were used to characterize each sample: methanol:ethyl acetate:water (2:2:1), methanol:ethyl acetate (1:1), and methanol:chloroform (1:1). Plates were visualized with iodine crystals and with UV light. All spots were noted and assigned Rf values. An anti-oxidant test was also conducted on the TLC plates using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) visualization solvent. The appearance of a fluorescent yellow spot on the TLC plate upon exposure to DPPH indicated the presence of free radical scavengers.
Cytotoxicity was evaluated with a brine shrimp assay. Two droplets of extract were added to vials of 50 shrimp and were counted after 1, 12, and 24 hours. The percent dead after each time increment was recorded.
Disk diffusion assays were conducted in the Cornell-Punta Cana Biodiversity Laboratory in the Dominican Republic. The bacteria Pseudomonas aeruginosa (gram-negative, ATCC #11778) and Bacillus cereus (gram-positive, ATCC #27853) as well the yeast Candida albicans (ATCC #90028) were streaked onto nutrient agar media (Difco 0001 for the bacteria and Difco 0382 for the yeast). Sterile disks were impregnated with 20 uL of plant extract, allowed to dry, and placed on the inoculated plates. Plates were incubated for 24 hours at 37( Celsius.
RESULTS AND DISCUSSION
The ten plants selected for this study are listed in Table 1according to their medicinal use by each midwife: maintenance of wellness during pregnancy, induction of labor or contractions, pain during childbirth, and postpartum infections of both mother and child.
Follow-up interviews with the midwives concerning the preparations and dosages of medicinal plants were inconsistent with initial interviews. It is important to note that the use of medicinal plants by midwives is not an exact science, and therefore difficult to quantify. It is recommended in the future to participate in the preparation of each remedy, as well as to conduct many follow-up interviews to obtain more accurate ethnobotanical data.
The preliminary chemical results from the TLC analysis indicate a wide range of compounds in the 10 species studied. Rf values are seen in the table below (Table 2).
Further investigation to determine which of these bands contribute to the biological activity of the plant is crucial to interpreting TLC data.
The characteristic chemistries of some of these plants has been previously studied. B. orellana contains carotenoid pigments in its orange-red seeds, and mono- and sesquiterpenes have been extracted from the leaves (Shultes and Raffauf 1990). G. barbadense and M. rudelara are both in the Malvaceae family which has a well-documented chemistry of flavonoids, cinnamic acids, tannins, ethereal oils, triterpenes, and occasional alkaloids. C. citratus contains terpenoid, essential oils. C. hediondinim contains steroidal saponins, sterols, catechin tannins, and flavonoids (Castner, et al. 1998, Shultes, et al. 1990).
Antioxidants were found in S. mombin, P. caimito, G. barbadense, M. rudelara, P. peltatum, C. hediondinim, and B. orellana. Further testing is needed to determine which of the compounds contributed to the free radical activity.
Results of the brine shrimp cytotoxicity assay are recorded in Table 3. High levels of cytotoxicity were exhibited by M. rudelara, B. orellana, and P. peltatum. These results proved interesting because both M. rudelara and B. orellana are used during pregnancy. Felicita recommended M. rudelara for pregnant women but only after five months of pregnancy. These results indicate that the cytotoxic effects of these plants may have some benefit in protecting the fetus from foreign pathogens.
High levels of cytotoxicity can also be good indicators of analgesic properties. The collected data that P. peltatum and B. orellana are both used to ease pain during childbirth support this evidence.
The results of the anti-bacterial and anti-fungal bioassays demonstrate growth inhibition (indicated by a clear zone of 2mm or greater) by B. orellana, P. caimito, S. mombin, and G. barbadense. High inhibition of Candida albicans was elicited by both P. peltatum and Z. officinalis. Bioassay results are listed in Table 4. P. caimito, S. mombin, and G. barbadense were all reported as treatments for bacterial and fungal infections of baby’s umbilical cord and the mother’s vagina after birth. B. orellana is administered as a tea during pregnancy, and may have some antibacterial benefits for the mother and child.
Bacterial data was assessed by (-) representing no activity and (+) representing >2mm inhibition. For the yeast bioassay, (-) also represented no activity and the active extracts had a range from (+1) 2 mm of inhibition, (+2) 4 mm inhibition, (+3) 10 mm inhibition, to (+4) equaling >12 mm inhibition.
In conclusion, the ethnobotanical information collected in this study demonstrates a preliminary classification of plants used by midwives in the Peruvian Amazon. It is a region of the world where childbirth is common, and where plants are the primary treatments of conditions during pregnancy and childbirth. Chemical analysis, biological assays, and ethnobotanical information serve as a basis for the scientific analysis of the treatments used. Midwives of the Peruvian Amazon have long been using these medicines, and we are just beginning to give scientific evidence to validate them.
REFERENCE
Brack Egg, Antonio. 1999. Diccionario Enciclopedico de Plantas Utiles del Peru. Cusco,
Perú: Centro de Estudios Regionales Andinos Bartolomé de las Casas.
Castner, J.L., Timme, S.L., and Duke, J.A. 1998. A Field Guide to Medicinal and Useful Plants of the Upper Amazon. Gainesville, FL: Feline Press.
Sáenz, Tula. 2000. Partos y Parteras: En la Cuenca del Río Marcará. Huaraz, Perú: Asociación Urpichallay.
Shultes, R.E. and Raffauf, R.F. 1990. The Healing Forest: Medicinal and Toxic Plants of the Northwest Amazonia. Portland, Oregon: Dioscorides Press.
______2000. Características Sociodemográficas de los Nacimientos en Lima Metropolitana. Lima, Perú: Instituto Nacional de Estadística e Informática.
______1996. Conducta Reproductiva y Maternidad en la Adolescencia en Lima Metropolitana. Lima, Perú: Instituto Nacional de Estadística e Informática.
______1992. Compendio Estadístico: Población Mujer y Salud, Region Loreto. Lima, Perú: Instituto Nacional de Estadística e Informática.
______1996. Comportamiento Reproductivo de Las Mujeres en las Comunidades Indigenas de la Amazonia Peruana. Lima, Perú: Instituto Nacional de Estadística e Informática,
______1999. Peru: Improving Health Care for the Poor. Washington, D.C.: The World Bank.
Peruvian women of the Amazon region have an average of 7.9 children, more than double the national average of 3.4 children. For these women, traditional methods of childbirth continue to be the primary means of healthcare. The collection and use of plants in the Peruvian Amazon by midwives is part of this tradition that has been sustained for centuries. The objective of this study is to evaluate natural products used during pregnancy and childbirth for their medicinal value. Structured interviews were conducted with three midwives in the communities of Jaldar and Jerusalen along the Yarapa River in the Loreto region of Peru. Ethnobotanical data was collected from the midwives and categorized into four treated conditions: maintenance of wellness during pregnancy, induction of labor or contractions, pain during childbirth, and postpartum infections of both mother and child. Ten plants common to each interview were identified and studied: Spondias mombin (Anacardiaceae), Pouteria caimito (Sapotaceae), Gossipium barbadense (Malvaceae), Gomphaena globosa (Amaranthaceae), Zingiber officinalis (Zingiberaceae), Malacra rudelara (Malvaceae), Piper peltatum (Piperaceae), Cymbogon citratus (Poaceae), Cestrum hediondinim (Solanaceae), and Bixa orellana (Bixaceae). Plants were extracted with 96% ethanol and chemical analysis was conducted with thin-layer chromatography (TLC). Preliminary results report anti-oxidant activity in Spondias mombin, Pouteria caimito, Gossipium barbadense, Malacra rudelara, Piper peltatum, Cestrum hediondinim and Bixa orellana. A brine shrimp assay indicated high levels of cytotoxicity in Bixa orellana, Malachra rudelara, and Piper peltatum. Biological activity was assessed using anti-bacterial and anti-fungal assays. Bacterial bioassays on Pseudomonas aeruginosa (gram-negative) and Bacillus cereus (gram-positive) revealed moderate growth inhibition by Bixa orellana, Pouteria caimito, Spondias mombin, and Gossipium barbadense. These four extracts also showed moderate growth inhibition of the yeast Candida albicans. High inhibition of Candida albicans was effected by both Piper peltatum and Zingiber officinalis.
There are two confounding factors that contribute to the high reproductive rate in the Amazon region of Peru. The first is the high number of children born to each woman in this region. These women have an average of 7.9 children, more than double the national average of 3.4 children (Compendium 1992). Particularly, in the Achual community of Jersalen where we studied, an average of 8.0 children are born to every woman (Compendium 1992). The second factor and one of the highest risks of reproduction in indigenous women is early childbearing. Among Amazonian girls ages 12-14, 2.3% are already mothers, a number nearly twice the national average. In adolescents ages 15-19, the number of mothers jumps to 34.4%, more than triple the percentage seen on a national level (Comportamiento 1996).
These statistics suggest the large role a midwife must perform in the community. Their work involves the collection and careful selection of herbal medicines used during pregnancy and childbirth. They determine the preparation in administering remedies to women in labor. Perhaps the most important role they perform is the passing on of knowledge of traditional medicines to others in their community (Sáenz 2000).
The objective of this study is to evaluate the natural products used by midwives and access their biological activity.
MATERIALS AND METHODS
Ethnobotanical data was collected through structured interviews with midwives in the communities of Jaldar and Jerusalen. Interviews were conducted with two midwives in Jaldar and one midwife in Jerusalen. Initially, each midwife was asked to describe specific plants used during pregnancy or childbirth. Further clarifications were made regarding dosage and preparation. In addition, voucher specimens were taken of each of the plants. A second follow-up interview was conducted with each midwife to specify exact uses and dosages.
Fresh green material was collected for each species studied. Leaves were ground and then extracted in 97% EtOH for 24 hours. Extracts were then filtered and dried using hot air to concentrate the samples.
A preliminary chemical analysis was performed using thin-layer chromatography (TLC). Extracts were spotted on aluminum-backed TLC plates coated with silica gel. Three solvent systems were used to characterize each sample: methanol:ethyl acetate:water (2:2:1), methanol:ethyl acetate (1:1), and methanol:chloroform (1:1). Plates were visualized with iodine crystals and with UV light. All spots were noted and assigned Rf values. An anti-oxidant test was also conducted on the TLC plates using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) visualization solvent. The appearance of a fluorescent yellow spot on the TLC plate upon exposure to DPPH indicated the presence of free radical scavengers.
Cytotoxicity was evaluated with a brine shrimp assay. Two droplets of extract were added to vials of 50 shrimp and were counted after 1, 12, and 24 hours. The percent dead after each time increment was recorded.
Disk diffusion assays were conducted in the Cornell-Punta Cana Biodiversity Laboratory in the Dominican Republic. The bacteria Pseudomonas aeruginosa (gram-negative, ATCC #11778) and Bacillus cereus (gram-positive, ATCC #27853) as well the yeast Candida albicans (ATCC #90028) were streaked onto nutrient agar media (Difco 0001 for the bacteria and Difco 0382 for the yeast). Sterile disks were impregnated with 20 uL of plant extract, allowed to dry, and placed on the inoculated plates. Plates were incubated for 24 hours at 37( Celsius.
RESULTS AND DISCUSSION
The ten plants selected for this study are listed in Table 1according to their medicinal use by each midwife: maintenance of wellness during pregnancy, induction of labor or contractions, pain during childbirth, and postpartum infections of both mother and child.
Follow-up interviews with the midwives concerning the preparations and dosages of medicinal plants were inconsistent with initial interviews. It is important to note that the use of medicinal plants by midwives is not an exact science, and therefore difficult to quantify. It is recommended in the future to participate in the preparation of each remedy, as well as to conduct many follow-up interviews to obtain more accurate ethnobotanical data.
The preliminary chemical results from the TLC analysis indicate a wide range of compounds in the 10 species studied. Rf values are seen in the table below (Table 2).
Further investigation to determine which of these bands contribute to the biological activity of the plant is crucial to interpreting TLC data.
The characteristic chemistries of some of these plants has been previously studied. B. orellana contains carotenoid pigments in its orange-red seeds, and mono- and sesquiterpenes have been extracted from the leaves (Shultes and Raffauf 1990). G. barbadense and M. rudelara are both in the Malvaceae family which has a well-documented chemistry of flavonoids, cinnamic acids, tannins, ethereal oils, triterpenes, and occasional alkaloids. C. citratus contains terpenoid, essential oils. C. hediondinim contains steroidal saponins, sterols, catechin tannins, and flavonoids (Castner, et al. 1998, Shultes, et al. 1990).
Antioxidants were found in S. mombin, P. caimito, G. barbadense, M. rudelara, P. peltatum, C. hediondinim, and B. orellana. Further testing is needed to determine which of the compounds contributed to the free radical activity.
Results of the brine shrimp cytotoxicity assay are recorded in Table 3. High levels of cytotoxicity were exhibited by M. rudelara, B. orellana, and P. peltatum. These results proved interesting because both M. rudelara and B. orellana are used during pregnancy. Felicita recommended M. rudelara for pregnant women but only after five months of pregnancy. These results indicate that the cytotoxic effects of these plants may have some benefit in protecting the fetus from foreign pathogens.
High levels of cytotoxicity can also be good indicators of analgesic properties. The collected data that P. peltatum and B. orellana are both used to ease pain during childbirth support this evidence.
The results of the anti-bacterial and anti-fungal bioassays demonstrate growth inhibition (indicated by a clear zone of 2mm or greater) by B. orellana, P. caimito, S. mombin, and G. barbadense. High inhibition of Candida albicans was elicited by both P. peltatum and Z. officinalis. Bioassay results are listed in Table 4. P. caimito, S. mombin, and G. barbadense were all reported as treatments for bacterial and fungal infections of baby’s umbilical cord and the mother’s vagina after birth. B. orellana is administered as a tea during pregnancy, and may have some antibacterial benefits for the mother and child.
Bacterial data was assessed by (-) representing no activity and (+) representing >2mm inhibition. For the yeast bioassay, (-) also represented no activity and the active extracts had a range from (+1) 2 mm of inhibition, (+2) 4 mm inhibition, (+3) 10 mm inhibition, to (+4) equaling >12 mm inhibition.
In conclusion, the ethnobotanical information collected in this study demonstrates a preliminary classification of plants used by midwives in the Peruvian Amazon. It is a region of the world where childbirth is common, and where plants are the primary treatments of conditions during pregnancy and childbirth. Chemical analysis, biological assays, and ethnobotanical information serve as a basis for the scientific analysis of the treatments used. Midwives of the Peruvian Amazon have long been using these medicines, and we are just beginning to give scientific evidence to validate them.
REFERENCE
Brack Egg, Antonio. 1999. Diccionario Enciclopedico de Plantas Utiles del Peru. Cusco,
Perú: Centro de Estudios Regionales Andinos Bartolomé de las Casas.
Castner, J.L., Timme, S.L., and Duke, J.A. 1998. A Field Guide to Medicinal and Useful Plants of the Upper Amazon. Gainesville, FL: Feline Press.
Sáenz, Tula. 2000. Partos y Parteras: En la Cuenca del Río Marcará. Huaraz, Perú: Asociación Urpichallay.
Shultes, R.E. and Raffauf, R.F. 1990. The Healing Forest: Medicinal and Toxic Plants of the Northwest Amazonia. Portland, Oregon: Dioscorides Press.
______2000. Características Sociodemográficas de los Nacimientos en Lima Metropolitana. Lima, Perú: Instituto Nacional de Estadística e Informática.
______1996. Conducta Reproductiva y Maternidad en la Adolescencia en Lima Metropolitana. Lima, Perú: Instituto Nacional de Estadística e Informática.
______1992. Compendio Estadístico: Población Mujer y Salud, Region Loreto. Lima, Perú: Instituto Nacional de Estadística e Informática.
______1996. Comportamiento Reproductivo de Las Mujeres en las Comunidades Indigenas de la Amazonia Peruana. Lima, Perú: Instituto Nacional de Estadística e Informática,
______1999. Peru: Improving Health Care for the Poor. Washington, D.C.: The World Bank.