Promising Potential of Lonchocarpus utilis against South American Myasis

Traditional medicine is especially important in the treatment of neglected tropical diseases because it is the way the majority of populations of affected countries manage primary healthcare. We present a case study that can serve as an example that can be replicated by others in the same situation. It is about the validation of a local remedy for myasis in Amazonian Ecuador, which is contrasted by bibliographic chemical reviews and in silico activity tests. We look for scientific arguments to demonstrate the reason for using extracts of Lonchocarpus utilis against south American myasis (tupe). We provide a summary of the isoflavonoids, prenylated flavonoids, chalcones, and stilbenes that justify the action. We make modeling predictions on the affinity of eight chemical components and enzyme targets using Swiss Target Prediction software. We conclude that the effects of this extract can be reasonably attributed to an effect of the parasite that causes the disease, similar to the one produced by synthetic drugs used by conventional medicine (e.g., Ivermectine).


Introduction
The World Health Organization (WHO) has estimated that more than 80% of the world's population routinely uses traditional medicine to meet their primary healthcare needs [1], and many traditional treatments involve the use of plant extracts or their active ingredients [2]. This is especially important in the treatment of neglected tropical diseases (NTD) [1]. The WHO has recognized twenty NTD: buruli ulcer, chagas disease, dengue and chikungunya, dracunculiasis (guinea-worm disease), echinococcosis, foodborne trematodiases, human african trypanosomiasis (sleeping sickness), leishmaniasis, leprosy (Hansen's disease), lymphatic filariasis, mycetoma, chromoblastomycosis and other deep mycoses, onchocerciasis (river blindness), rabies, scabies and other ectoparasites, schistosomiasis, soil-transmitted helminthiases, snakebite envenoming, taeniasis/cysticercosis, trachoma and yaws (endemic treponematoses). These diseases represent an important cause of morbidity, disability, and mortality in the poorest people living in developing countries. They are so named because of the lack of financial investment in the development of new drugs by high-income countries to treat them [3]. In this context, the Amazonian countries have large sectors of the population that use plants from the tropical forests where they live daily as a legacy of their ancestors. This represents knowledge that can be articulated with the Western scientist [4,5]. and

Cell
Inhibition of growth [36] 4-hydroxylonchocarpin Antifungal [56] Lonchocarpene Seedling Inhibition of growth/development [57]  As it can be observed in the probability graphics of these figures, rotenoids have shown the most affinity for ornithine decarboxylase (ODC), tyrosyl-DNA phosphodiesterase 1, arginine decarboxylase, NADH ubiquinone oxidoreductase chai-4, and the microtubule-associated protein tau (the latter especially for rotenone and rotenolone). Rotenone has shown the best relations with the cytochrome P450 group of enzymes.
Chalcones (4-hydroxylonchocarpin) have shown the most affinity with ODC, tyrosyl-DNA phosphodiesterase 1, arginine decarboxylase, and have a probability of more than 50% with different protein kinases.

Discussion
The hospital treatment of tupe is surgical: the application of local anaesthetics and removal of the larvae through the entrance orifice. For oral medical treatments, the drugs that might be employed are thiabendazole imidazols and macrocyclic lactones. Thiabendazole inhibits fumarate reductase, an enzyme specific for helminths. It is absorbed rapidly in the gastrointestinal tract, is metabolized in the liver, and it is eliminated by the kidney. However, it has many side effects. Ivermectin [58,59] has more selective activity with few systemic effects on mammals. It acts by binding to the anionic glutamate channels of gamma amino butyric acid (GABA) on the nerves and muscle cells of invertebrates, causing pharyngeal paralysis and death of the parasite by asphyxia and starvation. Our results for the Swiss Target prediction reports of isoflavonic rotenoids such as rotenone and rotenolone revealed strong interactions with many cytochrome P450 isozymes, NADH-ubiquinone oxidoreductase, and the microtubule-associated protein tau. Another rotenoid, deguelin, is especially akin to ornithine and arginine decarboxylases, and tephrosin shows affinity to tyrosyl-DNA phosphodiesterase, due to the probability levels shown in the Appendix A predictions. These results obtained by our in silico tests explain the activities of L. utilis. We also found a strong relation to the former decarboxylases and other groups of molecules of the extract: stilbenes (as lonchocarpene), chalcones (as 4-hydroxylonchocarpin), and prenylated flavones (such as 3 methoxylupinifolin). Rotenoids have the capacity to act against multiplication or growth, as summarized in the experimental results of Table 1. They cause a lack of energy, respiratory depression, respiratory arrest, and death. They lead to failure in the electron transport chain, which, at the mitochondrial level, translates into a blockade of the passage from ADP to ATP. Their inhibitory effect on NADHU-ubiquinone oxidoreductase has been demonstrated in the laboratory and as a consequence of the (ODC) phorbol ester-induced ornithine decarboxylase [60]. Rotenone is specifically classified as an insecticide type II with low toxicity to humans and warm-blooded animals [61]. The selective toxicity of rotenone in insects and fish versus mammals is related to its poor absorption from the gastrointestinal tract of the latter as well as the overall metabolic differences. Rotenone is converted into highly toxic metabolites in insects and fish. On the contrary, it is converted into non-toxic metabolites in mammals. In the motor system, 5-hydroxytriptamine (5HT) can depress GABA-mediated transmission and structures controlling movement [62]. Rotenone and rotenolone showed a great affinity for the membrane receptors of 5HT in our Swiss Target in silico tests, which is indirect evidence of the capacity of these molecules to behave similarly to Ivermectine when it causes helminth muscle paralysis.

Conclusions
In Latin America, myasis, as a disease, remains somewhat misunderstood. It is excluded from basic epidemiological research and hospital treatments are often lacking [63]. In this framework, traditional ethnomedicine is revealed as a powerful ally to improve the state of health, especially considering cases such as this case study. It can serve as an example that can be replicated by others in the same situation. We have looked for scientific arguments to demonstrate the reason for using extracts of Lonchocarpus utilis against south American myasis (tupe). We have provided a summary of the isoflavonoids, prenylated flavonoids, chalcones, and stilbenes that justify the action. We have performed modeling predictions on the affinity of eight chemical components and enzyme targets using Swiss Target prediction software.
We have concluded that the effects of this extract can be reasonably attributed to an effect of the parasite that causes the disease. Once again, the importance of the plant world in the drug discovery processes must be considered, and a call is made for plant conservation to be used as a source of obtaining added value bioproducts.

Ethnobotanical Study
All the information regarding the study where the data came from is available in Appendix B. It contains references to the voucher specimens of herbarium material collected, the permits and authorizations obtained, and the procedures applied. Tables A1 and A2 (in Appendix B) summarize the traditional uses given to the species.

Bibliographic Review
A bibliographic study was performed following the Prisma 2009 flow diagram methodology [64]. The databases accessed were Academic Search Complete, Agricola, Agris, Biosis, CABS, Cochrane, Cybertesis, Dialnet, Directory of Open Access Journals, Embase, Espacenet, Google Patents, Google Academics, Medline, PubMed, Science Direct, Scopus, Theseus, and ISI Web of Science. The aim was to find publications on chemical composition and/or activity. The abovementioned ones and the Latin names of the species and synonyms were used as keywords. The selected citations were summarized. Critical reading of this literature allowed us to elaborate on the discussion of the results and main statements.

In Silico Activity Test
Swiss Target prediction software [65,66] was eventually used to investigate the activity in silico to reinforce the principal arguments. Prediction reports were made with the following query molecules:  Appendix A Figure A1. Swiss Target prediction Report Files obtained using Rotenone as query molecule. Figure A1. Swiss Target prediction Report Files obtained using Rotenone as query molecule. Figure A2. Swiss Target prediction Report Files obtained using Rotenolone as query molecule. Figure A2. Swiss Target prediction Report Files obtained using Rotenolone as query molecule. Figure A3. Swiss Target prediction Report Files obtained using Deguelin as query molecule. Figure A3. Swiss Target prediction Report Files obtained using Deguelin as query molecule. Figure A4. Swiss Target prediction Report Files obtained using Thephrosin as query molecule. Figure A4. Swiss Target prediction Report Files obtained using Thephrosin as query molecule. Figure A5. Swiss Target prediction Report Files obtained using 3′ metoxylupinifolin as query molecule.   To perform the ethnobotanical survey under the Nagoya Protocol rules, collective written research consent was granted by the community president of the Assembly, Mrs. Luzmila Gayas. Prior individual consents had been obtained from the persons taking part in our survey. The survey consisted of a series of planned residential visits accompanied by Kichwa interpreters. Semi-structured interviews were recorded. Four knowledgeable elders of the Pakayaku community acted as informants and agreed to reveal their knowledge on the barbasku tree. The informants answered freely on several topics, including the common name of Kichwa, the part of the plant used, a description of its usage, the harvest season, storage (if any), preparation of concoctions, and the target of the treatment. After the fieldwork, the data were included in an spreadsheet Excel 2016 (Microsoft, Redmon, WA, USA). The existing ethnobotanical literature from Ecuador included in Table A1 was compared with the recorded uses that are summarized in Table A2.

Human Medicine
Used against myiasis: "to kill the tupe" (human bot fly) R Extraction of "milk" by pressure The "milk" is deposited on a piece of paper and placed where tupe has stung Hits and body aches R Crush roots Crushed root is placed directly on the skin

Veterinary
External antiparasitic R Extraction of "milk" by pressure The "milk" is deposited on a piece of paper and placed where tupe has stung

Catch fish R
Crushed roots to be used as soon as possible (in 1-2 days) The "milk" obtained is spread in the water of rivers and ravines