|Year : 2020 | Volume
| Issue : 2 | Page : 170-175
Comparison of the antimalarial activity of a Colombian traditional Uitoto remedy with laboratory preparations
Zully Johana Rodriguez1, Yinneth Victoria Rodríguez1, José Octavio García2, María Helena Arias1, Eric Deharo3, Giovanny Garavito1
1 Universidad Nacional de Colombia, Sede Bogotá, Facultad de Ciencias, Departamento de Farmacia, FaMeTra research group (traditional and Popular medicine Pharmacology), Colombia
2 Uitoto representative, Km 7 Via Leticia Tarapaca, Comunidad Ciudad Hitoma, Leticia, Colombia
3 Institut de Recherche pour le développement, IRD Représentation Ban Naxay, Saysettha District., Vientiane, Lao PDR
|Date of Submission||02-Feb-2019|
|Date of Acceptance||28-Feb-2020|
|Date of Web Publication||14-Jul-2021|
Dr Giovanny Garavito
Cra 30 No 45-03, Edificio 450, Lab. 306 Bogotá D.C. 111311
Source of Support: None, Conflict of Interest: None
Background & objectives: In Colombian Amazonia, Uitoto indigenous people use a preparation of Curarea toxicofera (Menispermaceae) to prevent and treat malaria. To open the way for the production of a standardized herbal remedy, we compared the activity of the traditional preparation with laboratory preparations.
Methods: People were interviewed on their mode of use and preparation of what is considered the best remedy against fevers in this area. The herbal remedy was prepared according to the healer’s recommendations. The plant was also submitted to continuous distillation and percolation extraction. The preparations were then tested against Plasmodium falciparum, in vitro. Traditional preparation and extract obtained by percolation were tested on Plasmodium berghei infected mice. Chemical profiles were also explored by thin-layer chromatography.
Results: Yields of extraction were around 7% in the preparations (percolation was the most efficient). The phytochemical profile showed a mix of steroids, flavonoids and alkaloids qualitatively similar in all preparations. In vitro, the extracts showed inhibitory concentration 50 <10μg/mL: the traditional preparation was almost three times less active than laboratory preparations. In vivo, percolation was also more active than traditional preparation, inhibiting 78% of the parasite growth at 400mg/kg/day by oral route.
Interpretation & conclusion: Pharmacological activities suggest that both the original remedy (prepared according to traditional pharmacopeia) and the extracts obtained by percolation extraction exhibit relevant antiparasitic activity. C. toxicofera should therefore be considered for the elaboration of an improved traditional medicine by implementing toxicological studies and carefully following quality control guidelines for its preparation.
Keywords: Antimalarials; Curarea toxicofera; ethnopharmacology; traditional medicine, improved traditional medicine
|How to cite this article:|
Rodriguez ZJ, Rodríguez YV, García JO, Arias MH, Deharo E, Garavito G. Comparison of the antimalarial activity of a Colombian traditional Uitoto remedy with laboratory preparations. J Vector Borne Dis 2020;57:170-5
|How to cite this URL:|
Rodriguez ZJ, Rodríguez YV, García JO, Arias MH, Deharo E, Garavito G. Comparison of the antimalarial activity of a Colombian traditional Uitoto remedy with laboratory preparations. J Vector Borne Dis [serial online] 2020 [cited 2021 Aug 5];57:170-5. Available from: https://www.jvbd.org/text.asp?2020/57/2/170/310868
| Introduction|| |
Malaria is a life-threatening parasitic disease spreading all over tropical regions; in 2016, 216 million new cases and more than 445,000 deaths were reported worldwide. In South America, the disease is mainly concentrated in Brazil, Venezuela and Colombia. Around 85% of the territory of Colombia has optimal conditions for the survival of the vector and disease transmission, which is of great concern for health authorities with 134,400 cases reported in 2016, nearly double the number reported in 2015 (74,100 cases).
In Colombia, 87 indigenous ethnic groups have been recorded, mainly in the provinces of Vaupés, Guainía, Guajira, Amazonas, Vichada, Cauca, and Nariño. The members of the Jitoma (Ziora Amena) community (meaning “man of the river”) are located in the province of Amazonas and are thought to have descended from the Muiname (linguistically Uitoto, Huitoto or Witoto) ethnic group, who use the Bora language. The Uitoto oral culture contains complex narratives, describing the living conditions in their “Maloca” (traditional cacique family house), detailing the way they play the “manguaré” (instrument used as a means of communication) and highlighting their admiration and respect for nature.
The “Abuelo” (grandfather) Don José Octavio García, also known by his traditional name “Jitoma Safiama” (sunlight), is the medicine man of the community and has inherited the knowledge of his predecessors: Don José García, his father, who was trained as a healer by his grandfather Kima Baiji, and recognised as the great Shaman of the Amazonian Colombo-Peruvian Muinane nation; he mentioned that many of the plants used to treat malaria “can be poison or remedy at the same time” depending on dosage and combination and that the treatment should be concomitant to ritual practice. The perfect place to do these rituals is the “Maloca”, where people meet to exchange all kinds of information. Knowledge about plants is also acquired through meditation and consumption of “mambe” to facilitate communication with jungle spirits. “Mambe” is obtained by mixing roasted and pulverized Erythroxylon coca L. leaves with the ash of Pourouma cecropiifolia Mart. leaves. According to Uitotos, malaria is a disease “that came with the white people” and for which they did not previously have any remedies. They then had to find medicines in the forest. It is described as “the disease of evil, occurring when mosquitoes bite”. They also describe symptoms such as “you become yellow, your head is heavy, you do not eat, you feel heat in the chest and in the stomach, you are very hot, you have cold and dry sweat, after sweating the skin turns dry, you feel very cold, you are restless and feel desperate”. The “grandfather” (Jitoma Safiama), was able to differentiate between periodic malaria fevers and others; he was also able to identify the mosquito responsible for transmission.
Previously, the use of plants was evaluated for the treatment of malaria by the community, with five traditional preparations, (among three plants: Aspidosperma excelsum, Curarea toxicofera and Abuta grandifolia) with C. toxicofera being ten times more active in vitro against P. falciparum FCB-2 and the only one that, in vivo, against P. berghei ANKA, generated a significantly lower parasitemia compared to the infection control group. Also, we studied in vitro and in vivo antiplasmodial activity of traditional preparations of C. toxicofera alone and in combination with classical antimalarials and we showed that it was a plant of interest for further investigation.
Indeed, in Colombia, although access to conventional treatments is a part of national health programs, the use of traditional medicine is still common,. Nevertheless the quality of a traditional remedy is highly variable, which could affect safety and efficacy. It is clear that quality control should be a key concern for the Colombian herbal system. Standardization should be implemented, from the source of plant origin all the way to pharmacological profiling of the finished product. In this context, our goal was to elaborate a traditional preparation in laboratory conditions and to compare it to a preparation made in the field. For that purpose, we explored the antimalarial activity of preparations of C. toxicofera, and determined their phytochemical profiles in order to open the way for the elaboration of an improved traditional medicine under quality assurance guidelines.
| Material & Methods|| |
The Uitoto community and the Universidad Nacional de Colombia have been working together for more than a decade. People collaborate actively with researchers and students in the frame of agreements following the convention on biological diversity and Nagoya protocol guidelines, including benefit sharing agreements and informed consent. The present study respects administrative aspects of the ANLA (National Environmental Licensing Authority) resolution No. 0255-12/03/2014 and resolution No. 1505-2019 (contracts for access to genetic resources and derived products No. 269), Ministry of Environment and Sustainable Development.
Plant collection and extracts preparation
Curarea Toxicofera (Wedd.) Barneby & Krukoff, (Colombian origin) was collected in the municipality of Leticia-Amazon, at GPS location S 04° 07. 768’ W 069° 55. 246. Avoucher specimen has been deposited at the Colombian National Herbarium (voucher N°: COL 570486). A specialist identified all collected material and the plant name has been checked with The Plant List (https://www. theplantlist.org/).
Extracts were prepared as follows: in the field, the traditional remedy was prepared by one of the traditional healers of the indigenous community, it involved cleaning the fresh liana, cutting it into pieces (about 5 cm wide) and preparing a decoction of around 70 g in water (2L) on wood fire for approximately one hour. The end point of the cooking was determined by the flavour and the colour, which should become red, but the healer told us that he must also taste it to define “the final point” when 80% of the water has evaporated, he also added that the preparation must be accompanied by evocations of supernatural forces in the presence of the patient, as the treatment is specific to the person; this preparation was named “COM”.
Continuous reflux distillation (CD) was applied to small pieces of plant placed in a glass flask with water and exposed to heat for one hour (by condensing vapours into the flask); in the percolation extraction (PE), cold water was poured on the pulverized plant until exhaustion (monitored by thin-layer chromatography). A final volume of 500 mL was then filtered and lyophilized (Labconco Freezone 4.5). All preparations were stored at 4°C, in an environment protected from daylight, until use.
Preliminary phytochemical analysis
Thin-layer chromatography (TLC) on Silicagel 60G F254 and elution solvent such as ethyl acetate, methanol, acetic acid were used to assess the presence of metabolites with UV light at 254 and 365 nm after spraying with different types of reagents (Dragendorff, NP/PEG, Liebermann-Burchard) according to Wagner.
Cultures of the FCR-3 chloroquine resistant strain of P. falciparum (donated by the Malaria Research Group of the University of Antioquia) were carried out according to Trager and Jensen, on glucose-enriched RPMI 1640 medium supplemented with HEPES and 10% human serum at 37°C. The tests were conducted according to Deharo and adapted from Garavito in 96 flat-bottom well plates. Each well, containing 0.1 ml of a previously synchronized culture with ring stage predominantly (2.5% hematocrit, 2% parasitaemia) was exposed to increased concentration of the test compounds (lyophilized) ranging from 0.78 to 100 μg/ml. Growth control wells received 0.9% saline solution and active control wells, chloroquine (Sigma Lot 84H1169). The impact of the extracts on parasite growth was measured by optical microscopy (Nikon Eclipse) on smears stained with giemsa. Almost 10,000 red blood cells were counted for each concentration.
The percentage of growth inhibition of P. falciparum was calculated by the following formula.
The inhibitory concentration 50 (IC50) was determined by regression analysis using the GraphPad Prism 6 software.
Antimalarial activity in vivo
The in vivo antimalarial activity of plants extracts were determined by the classical 4-days suppressive test against Plasmodium berghei ANKA and adapted from Garavito. Studies were conducted in accordance with the internationally accepted principles for laboratory animal use, specifically to the French and Colombian guidelines on laboratory animal use and care (N° 2001-464 and N° 008430) and approved by the institutional ethical committee.
Swiss mice, of a mean body weight 20+/-2 g, with food and water ad libitum, were infected with 10 parasitized cells in 0.9% saline, on Day 0. Groups of 5 mice were orally treated from Day 0 to Day 3 with 400 mg/ kg/day of COM and PE extracts. The active control was chloroquine.
The suppressive effect was estimated on Day 4, examining giemsa-stained thin blood smears made from the tail of the treated mice and compared with infection control group of mice treated with the solvent of the extract (water). The stained thin blood smears were examined under x1000 magnification, and the percentage of parasitized red blood cells was counted on at least 9,000 red blood cells observed for each concentration. Percentage growth inhibition of the parasite was calculated by the following formula.
The activity was categorized according to Willcox et al.
A 2% red blood cell suspension was prepared in PBS. Aliquots of 100μl were distributed in a 96-well plate, extracts were added at doses ranging from 0.001 to 100 μg/ml. Positive control was Triton X-100 at 0.1 % while negative control was PBS. Plates were incubated at 37°C for one hour, then centrifuged 500G 10 minutes. The supernatant was transferred into another flat bottom 96 wells plate and read at 418 nm. The percent of haemolysis was calculated as follow.
HepG2 and MRC5 cells (human liver cancer cells and human fetal lung fibroblast cells, respectively) were seeded (150,000 cells/ml, 100μl/well) in a 96-well flat-bottom plate at 37 °C and with 5% CO2 RPMI 1640 supplemented with 10% heat-inactivated foetal bovine serum. Compounds were added at varying concentrations and the cells were cultured for 48 h. The effect was determined using a classical resazurin viability assay (0.15 mg/ml in PBS). Optical density was read at 540 nm and 600 nm, excitation and emission wavelengths respectively, with a 96-well fluorescence scanner (Twinkle LB 970 Berthold Technologies). All experiments were performed in triplicate. The CC50 was determined by regression analysis.
ANOVA (Microsoft Excel 2010) and the Tukey test were performed. Significance was set up at p <0.05.
| Results|| |
We used water extraction, as it was the most similar method to the original preparation. The final extraction technique was chosen according to practical, convenient and reproducible criteria. Percolation extraction showed a better yield of extraction (8.9%) than continuous distillation (7.3%) and traditional preparation (approx. 6.9%). This factor must be taken into account when scaling up the process for a semi-industrial setting.
By thin layer chromatography it was revealed the presence of alkaloids, flavonoids, steroids and polyphenol [Table 1].
|Table 1: Qualitative phytochemical characteristics of the extracts by thin-layer chromatography|
Click here to view
Biological activity and toxicity
In vitro antiplasmodial activity and antimalarial activity of the preparations are shown in [Table 2]. There was no haemolysis even at 100 μg/mL, the maximal concentration tested, also, there was no detectable toxicity at concentrations <50μg/mL either on HepG2 or MRC5 cells.
|Table 2: IC50 of the preparations on P. falciparum FCR-3 strain and percentage of inhibition on P. berghei strain in vivo|
Click here to view
| Discussion|| |
During the survey in the forest, it was evident that the choice of certain species was driven by aroma and flavour, according to the healer “the more bitter the plant, the higher the content of quina, and the more efficient it is”. Interestingly, the correlation between bitterness and activity is a trans-Amazonian and a trans-continental belief as this has been described in Saramaka people in Suriname as well as among Bunong people in Cambodia.
According to the healer, one of the most effective plant was Curarea toxicofera (Wedd.) Barneby & Krukoff. This is a Menispermaceae, native to Andean lowlands, found in the rainforest and distributed across Bolivia, Brazil, Peru, and Colombia. According to The Plant List, C. toxicofera has many botanical synonyms: Abuta boliviana Rusby, Chondrodendron bioccai Lusina, Chondrodendron polyanthum (Diels) Diels, Chondrodendron toxicoferum (Wedd.) Krukoff & Moldenke, Cocculus toxicoferus Wedd., Hyperbaena polyantha Diels. Among these mentioned plants, only Abuta boliviana has been reported as a “cure-all” plant. The infusion of leaves is supposed to be diuretic, carminative, useful for difficult digestion, menstruation, uterine cramps, rheumatism, and liver disorder,,.
The preparation of herbal remedies in laboratory conditions, preserving pharmacological efficacy and safety, depends on many factors including the quality of raw material, the technique of extraction and the drying and grinding processes to limit alteration of active metabolites. According to Valencia et al. it is likely that extraction is more influenced by the type of solvent than by the method used for this purpose.
Thin layer chromatography is a simple tool accessible in non up-to-date laboratories in developing countries to follow up the reproducibility of the extraction technique. It revealed the presence of alkaloids, flavonoids, steroids, and polyphenol [Table 1]. Interestingly, all preparations showed the same profile, with predominance of alkaloids, as also reported elsewhere for the Curarea genera,,. No cardiac glycosides or saponins were detected.
All three forms of preparation (PE, CD, and COM) exhibited good to moderate activity in vitro, according to the criteria defined by the network “Research Initiative on Traditional Antimalarials” (RITAM). The preparations made in the laboratory (IC50 around 2 μg /ml) were almost 3 times more active than the traditional preparation (IC50 around 6 μg/ml) (p <0.05; [Table 2]) and 5 times less active than chloroquine (p <0.05), the pure reference compound. Notably, there was no difference between the results obtained with our traditional preparation made from plants collected in the dry season and the results reported previously by our team with plants collected during the rainy season. The activity was not related to any haemolytic effect as there was no haemolysis even at 100 μg/mL, the maximal concentration tested.
The 4-days test on P. berghei infected mice showed that both preparations (COM and PE) were able to reduce more than 60% of the parasite growth at 400 mg/kg/ day per os, generating significantly lower parasitemias when compared to infection control group (p <0.05). The laboratory preparation was slightly more active than the traditionally prepared remedy. There was no difference between preparations in mean survival time (p = 0.68). No sign of toxicity was observed on the animal; the animal returning to normal activity after treatment. Nevertheless, anatomopathology study should be conducted.
Developing a new medicine from natural products could be an important source of new antimalarials but it is a lengthy and very expensive process. The development of standardized and validated phytomedicines could be a more rapid and economic way to propose efficient alternative treatments. This is the case in Mali where the government adopted policies regulating extracts but not crude traditional preparations. According to a World Health Organisation (WHO) statement: “If the product has been traditionally used without demonstrated harm, no specific restrictive regulatory action should be undertaken unless new evidence demands a revised risk–benefit assessment”.
| Conclusion|| |
C. toxicofera collection, preparation, method of administration and safety are already understood in traditional Uitoto knowledge and health care. WHO states that there is no requirement for preclinical toxicity testing, but rather the evidence of traditional use or recent clinical experience is sufficient. However, we suggest that in the case of a C. toxifera improved phytomedicine, a preclinical trial should be set up in different models, to detect any toxicity. This study was able to reproduce the traditional remedy in laboratory conditions while maintaining its activity, evidencing the presence of alkaloids and sheds light on the most convenient extraction method.
Conflict of interest: None
| Acknowledgements|| |
The authors would like to thank to the Jitoma (Ziora Amena) community, especially to Jesus Garcia, for friendship, availability and their collaboration during many years of fieldwork together. ZJ Rodríguez and YV Rodríguez are grateful to Departamento Administrativo de Ciencia, Tecnología e Innovación (Colciencias) for their MSc scholarships (Convocatoria 617 de 2013, Jovenes investigadores e innovadores), MH Arias is grateful to Colciencias for their Ph.D. scholarship (Convocatoria 647, Doctorados Nacionales 2014) and this work was supported by the Vicedecanatura de Investigación, Facultad de Ciencias, Universidad Nacional de Colombia. Authors are also grateful to Jorge E. Hernandez for phytochemical advice and Elizabeth Elliott for editing the manuscript.
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[Table 1], [Table 2]