Amazonian plants from ethnomedicine to biotechnology through pharmaceutical biology approaches: a PhD experience in connecting forest with laboratory

The South american Natives, Shuar and Achuar people and their ethnomedical culture constitute the background subject of the Phd research, performed both in Ecuador (Salesian Politechnic University, Quito), and in Italy (Pharmaceutical biology labs, University of Ferrara). Based on ethnomedical responses, Piper aduncum, Maytenus macrocarpa, Schinus molle, Tecoma stans and Eugenia hallii were chosen as amazonian plant species subject of the research. AIMS The research has been focused on: − checking the presence of endophytic fungi in plants; − isolating and subculturing pure endophytic strains; − checking the biotransformation capacity of the isolated endophytes on pure compounds; the most performing endophytes were also tested on phytocomplexes and pure chemicals obtained by the plant from which the fungi were isolated; − phytochemical characterization and bioactivity assays of plant extracts: P. aduncum. − METHODS Biotransformations. Fresh aerial plant parts were properly washed in sanitizing solutions and in vitro cultured using adequate solid media to isolate endophytes. (+/-)-cis-bicyclo[3.2.0]hept-2-en-6-one, acetophenone, 1-indanone, 2-furyl methyl ketone, 2-methylcyclopentanone, 2-methylcyclohexanone, 2- methoxycyclohexanone were chosen as substrate model for biotransformations. The cultures were sampled after 1, 3, 7, 10 days of culturing, and ethyl acetate extracted to verify by GC-MS the presence of possible biotransformation products. Biotransformations were also checked on P. aduncum whole essential oil and on dillapiol, cis-ocimene, piperitone, (-)-terpinen-4-ol as most abundant chemicals. Chemical fingerprinting of P. aduncum essential oil. Steam distillation was adopted to obtain the essential oil, then characterized by GC-MS, NMR analyses. In vitro bioassays of P. aduncum essential oil. Antimicrobial activities were checked in vitro using proper agarized media to reach MIC. Antioxidant capacities were checked through DPPH test, ABTS and photochemiluminescence assays. Born's turbidimetric method and Writhing test were respectively adopted to check platelet-aggregation and anti-nociceptive properties. Mutagenic, antimutagenic properties and toxicity were assayed using classical and modified Ames test. MAIN RESULTS 364 fungal strains were in vitro isolated. Among all, 5 strains performed biotransformations on acetophenone to (S)-1-phenylethanol, with important yields (78-97%) and enantiomeric excess (78- 100%). Three strains gave also phenols probably by enzymatic reactions (Baeyer-Villiger oxidations). 15 fungal strains gave the lactones (-)-(1S,5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one and (-)-(1R,5S)-3- oxabicyclo[3.3.0]oct-6-en-2-one from (+/-)-cis-bicyclo[3.2.0]hept-2-en-6-one, probably as result of monooxygenase activation. Phytochemical characterization of P. aduncum essential oil has evidenced dillapiol as the most abundant terpene, followed by cis-ocimene, piperitone and terpinen-4-ol. Only cisocimene and piperitone gave several biotransformation products through dehydrogenation and hydroxylation reactions. The essential oil has evidenced non-mutagenic properties and interesting antifungal and antioxidant activities. CONCLUSIONS Several endophytic fungal strains from Amazonian plants were isolated and checked for biotransformations on pure chemicals and on P. aduncum essential oil. Data obtained will be useful for possible following patents about micro-organisms able to transform pharmaceutically interesting chemicals. Taxonomical characterization of the most performing fungal strains is still in progress. P. aduncum essential oil can be considered genotoxically safe and provides interesting antifungal and antioxidant properties, supporting its ethnomedical use as cicatrising and disinfectant crude drug and suggesting an extension of its employ as preservative ingredient

Petroleum Degradation: Promising Biotechnological Tools for Bioremediation

One of the most common chemicals involved in the soil contamination or soil pollution is petroleum hydrocarbons (PHs). As we know that PH-contaminated soil affects human health directly, such as (i) contact with soil, (ii) via inhalation of vaporized contaminants, and (iii) infiltration of soil contamination into groundwater aquifers used for human consumption. Microbiological processes play an important role in the removal of PHs and take advantage of the catabolic versatility of these organisms to degrade such compounds either partially or completely (mineralization). Thus, the present chapter moves around the relationship of microorganisms with PHs. Based on this concept, this chapter has been designed to address the following relevant issues: How to isolate PH-degrading microorganisms by co-enrichment and optimized enrichment methods? How to study the microbial community structure by high-throughput sequencing method? What are the metabolic versatilities of microorganisms for degrading PHs? How to treat the environmental problems through biological means? What are the available ecotoxicity studies for the analysis of residual PHs after the microbiological treatment at the PHs-contaminated sites? Thus, the aim of this chapter is to explain the importance of microorganisms in cleaning the oil-contaminated environments

Etnofarmacología, bioactividad y fitoquímica de Maxillaria densa Lindl. Revisión científica y biocomercio en el neotrópico

Maxillaria densa is a native American orchid extended in high mountains and humid forests. The objective of this review is to provide a critical analysis on ethnopharmacology, phytochemistry and pharmacology of the species, focusing on its potential for biotrading. The study was conducted exploring the PubMed, Scopus, SciELO and SciFinder databases and specialized websites, looking for bioactive molecules and thus confirming what properties and compounds contain this plant. M. densa is traditionally used for the treatment of stomach pains, such as antidiarrheal and antispasmodic. The extracts obtained from M. densa show the presence of six active ingredients derived from phenanthrene, an aromatic polycyclic hydrocarbon. The biological activity studies carried out have shown spasmolytic, antinociceptive and relaxing effects. M. densa grows in coffee crops under shade, which are considered sustainable production systems, increasing the chances of green businesses for local farmers. From the mentioned findings, M. densa presents a potential as a source of bioactive molecules and as an ornamental flower, opening a new perspective in the Bioeconomy field.Maxillaria densa es una orquídea oriunda de América Central extendida en alta montaña y bosques húmedos. El objetivo de esta revisión es proporcionar un análisis crítico sobre etnofarmacología, fitoquímica y farmacología de la especie, enfocándose en su potencial para el biocomercio. Para encontrar sus compuestos bioactivos fueron exploradas las bases de datos de PubMed, Scopus, SciELO y SciFinder, Sciencedirect, Springer, la editorial Elsevier y webs especializadas, de esta manera se confirmó cuáles son los compuestos y propiedades de esta planta. M. densa es usada tradicionalmente para el tratamiento de dolores estomacales, como antidiarreico y antiespasmódico. Los extractos obtenidos de M. densa muestran la presencia de seis principios activos derivados del fenantreno, un hidrocarburo policíclico aromático. Los estudios de actividad biológica realizados han evidenciado efectos espasmolíticos, antinociceptivos y relajantes. M. densa crece en cultivos de café bajo sombra, que son considerados sistemas de producción sostenibles, aumentando las posibilidades de negocios verdes por agricultores locales. A partir de los hallazgos mencionados, M. densa presenta un potencial como fuente de moléculas bioactivas y como flor ornamental, abriendo una nueva perspectiva en el ámbito de la bioeconomía

Biological and chemo-diverse characterization of Amazonian (Ecuador) Citrus petitgrains

Six Amazonian petitgrains samples from C. nobilis Lour., C. aurantium L., C. limon L. and mixture of Citrus spp.(Rutaceae), named CN, CA, CL1, CL2, C1 and C2, were chemically characterized by GC-MS and 13C NMR and evaluated for antioxidant acitivity (DPPH and b-carotene bleaching tests), for antimicrobial properties (disk diffusion method) and for antifungal capacity (agar vapour assay). CN, C1, C2 samples evidenced the most interesting results: CN (g-terpinene/linalool chemotype: 14.3%/41.6%, with a considerable amount of thymol: 9.0%), and C1 (linalool, 18.3%; sabinene, 11.6%; thymol, 5.5%), showed relevant antioxidant activity with both DPPH (IC50=3.52 and 5.48 mg/ml, respectively) and b-carotene (IC50=0.387and 0.491 mg/ml, respectively). Antibacterial properties of CN and C1 against P. mirabilis (MIC=0.61 mg/ml for both)and B. subtilis (MIC=0.61 and 0.44 mg/ml, respectively) were most probably due to thymol.C2 (geranial: 34.7%, neral: 33.1%) evidenced a valuable bioactivity against Candida albicans (MIC=0.44 mg/ml).The 50% growth inhibition (IC50) of the dermatophytes T. mentagrophytes and N. cajetani was reached with amounts ofC1, C2 and CN less than 4 ml/plate. Bioactivity of Amazonian Citrus spp. CN, C1 and C2 essential oils suggests their potential use as food preservatives or additives in cosmeceuticals as preventive against dermatophytic fungal infections

Productos naturales: investigación y perspectivas en Ecuador

Ecuador es un país privilegiado por su riqueza en organismos vivos íntimamente relacionados con su alta biodiversidad. En los últimos 20 años, las universidades públicas y privadas, así como los institutos de investigación, han invertido en la formación de sus docentes-investigadores como en tecnología de última generación relacionada con este tema. Este texto refleja los avances en la producción científica que se han generado en el país. Tres capítulos son revisiones exhaustivas del uso tradicional y el potencial de las plantas medicinales en problemas de salud y aplicaciones puntuales. Un capítulo analiza las plantas medicinales y sus metabolitos en una determinada área biogeográfica del Ecuador. Una revisión se centra en el aceite esencial de una especie nativa de elevado potencial bio-económico como la Psidium guajava. Dos estudios experimentales confirman la riqueza fitoquímica y el potencial medicinal de especies de nuestra biodiversidad como la Persea americana y Chuquiraga jussiuei. Y finalmente, un capítulo analiza la importancia del control en los productos naturales procesados con base en su calidad microbiológica y la relación con las correctas prácticas de manufactura. Este texto confirma que Ecuador tiene mucho que mostrar al mundo de la ciencia

Biotransformations by endophytic fungi isolated from traditional Ecuadorian medicinal plants: Connecting ethnomedicine with biotechnology

Ecuador, a small country with diverse ecosystems in the Amazon, Andes and Pacific coastal regions is considered one of the 17 "megadiverse” countries, and the native ethnic groups and rural communities have a strong ethnomedical tradition in the use of native plants in healing. Traditional ethnobotanical knowledge can be used to guide biotechnological research on medicinal plants, even when the new application is an innovation only distantly related to the traditional use. Based on ethnomedicalknowledge of indigenous communities, the following plants from the Amazon and Andes regions were chosen for investigation: Piper aduncum (Piperaceae), Maytenus macrocarpa (Celastraceae), Schinus molle (Anacardiaceae), Tecoma stans (Bignoniaceae) and Myrcianthes hallii (Myrtaceae). The research was focused on (i) assesing the presence of endophytic fungi in the selected plants, (ii) isolating and subculturing in vitro pure endophytic strains, (iii) assessing the biotransformation capacity of the isolated endophytes on pure compounds (intermediates of pharmaceutical synthesis). The following compounds were chosen as substrate models for biotransformations: (+/-)-cis-bicyclo[3.2.0]hept-2-en-6-one, acetophenone, 1-indanone, 2-furyl methyl ketone, 2-methylcyclopentanone, 2- methylcyclohexanone, 2-methoxycyclohexanone. A total of 364 fungal strains were isolated in vitro; among these, five strains performed biotransformations on acetophenone to (S)-1-phenylethanol, with important yields (78-97%) and enantiomeric excess (78-100%). Three strains also yielded phenols, probably by enzymatic reactions (Baeyer-Villiger oxidations). Fifteen fungal strains yielded the lactones (-)-(1S,5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one and (-)-(1R,5S)-3- oxabicyclo[3.3.0]oct-6-en-2-one from (+/-)-cis-bicyclo[3.2.0]hept-2-en-6-one, probably as result of monooxygenase activation.

ACTIVIDAD ANTIF aNGICA in vitro DE ACEITES ESENCIALES DE Ocotea quixos (Lam.) Kosterm.

Los hongos fitopat\uf3genos ocasionan relevantes p\ue9rdidas de los cultivos agr\uedcolas, tanto en fase de campo como en poscosecha. Se evalu\uf3 el uso de aceites esenciales extra\ueddos de plantas amaz\uf3nicas, en diferentes concentraciones, para el control in vitro de estos hongos. Los aceites esenciales se obtuvieron por destilaci\uf3n en arrastre de vapor a partir de dos especies vegetales (Ocotea quixos y Piper aduncum) colectadas en la provincia amaz\uf3nica de Pastaza, Ecuador. La actividad antif\ufangica se evalu\uf3 mediante el m\ue9todo de la difusi\uf3n en agar sobre los hongos fitopat\uf3genos Aspergillus oryzae, Cladosporium cladosporioides, Fusarium solani, Rhyzopus stolonifer, Moniliophthora roreri y Phytophthora sp. El aceite esencial de O. quixos usado en su m\ue1xima concentraci\uf3n (500 \ub5L\ub7mL-1) inhibi\uf3 el crecimiento de todas las cepas alcanzando 94 % de inhibici\uf3n promedio y registrando un comportamiento similar al aceite esencial de tomillo (Thymus vulgaris), el cual fue utilizado como patr\uf3n de comparaci\uf3n. El aceite esencial de P. aduncum, tambi\ue9n en su m\ue1xima concentraci\uf3n mostr\uf3 los mayores porcentajes de inhibici\uf3n frente a F. solani (94 %) y Phytophthora sp. (91 %). Los resultados sugieren el empleo de estos aceites esenciales como agentes de control biol\uf3gico de hongos fitopat\uf3genos. Palabras clave adicionales: Actividad biol\uf3gica, pat\uf3genos vegetales, plantas amaz\uf3nicas ABSTRACT In vitro antifungal activity of essential oils of Ocotea quixos (Lam.) Kosterm. and Piper aduncum L. The phytopathogenic fungi cause relevant crop losses, both under field and post-harvest conditions. The use of essential oils extracted from Amazonian plants, at different concentrations, was investigated, in order to evaluate in vitro properties against those fungi. Essential oils were obtained by steam distillation, from two plant species (Ocotea quixos and Piper aduncum) collected in the Amazonian province of Pastaza, Ecuador. The antifungal activity was evaluated through the agar diffusion method against the Aspergillus oryzae, Cladosporium cladosporioides, Fusarium solani, Rhyzopus stolonifer, Moniliophthora roreri and Phytophthora sp. The O. quixos essential oil, in its maximum concentration (500 \ub5L\ub7mL-1), inhibited the growth of all strains, reaching an average of 94 % inhibition rate, similar to the essential oil of thyme (Thymus vulgaris), which was used as standard. The P. aduncum essential oil, also in the maximum concentration, showed the best inhibition percentage against F. solani (94 %) and Phytophthora sp. (91 %). These results suggest the use of both essential oils as agents of biological control of phytopathogenic fungi. Additional key words: Amazonian plants, biological activity, phytopathogens <BR

Assessing Ecosystem Services Supplied by Agroecosystems in Mediterranean Europe: A Literature Review

Agricultural landscapes in the Mediterranean region may be considered as social-ecological systems that are important for biodiversity conservation whilst contributing to a wide range of ecosystem services. This literature review aims to identify the current state and biases of ecosystem service assessment in agroecosystems within the Mediterranean region, evaluate pressures impacting on agroecosystems and their services, and practices that promote ecosystem service synergies in Mediterranean agroecosystems. A total of 41 papers were selected for analysis from a set of 573 potentially relevant papers. Most of the selected papers focused on supporting, regulating and provisioning services, and mostly assessed ecosystem structure or services in the European Mediterranean context. Literature about benefits and values ascribed to by communities and stakeholders remain limited. Results presented here support the notion of multifunctional Mediterranean agroecosystems and multiple synergies were recorded in this review. Publications dealing with pressures that related to agricultural practices and demographic changes were in the majority and impact on different cropping systems. This review highlights the need to carry out integrated ecosystem service assessments that consider the multiple benefits derived from agroecosystems and which may be used to identify management practices that lead to the improvement of ecosystem services capacities and flows

Cytotoxic Effect and TLC Bioautography-Guided Approach to Detect Health Properties of Amazonian Hedyosmum sprucei Essential Oil

Bioautography has been used as rapid and easy strategy to detect and identify bioactive fractions/molecules in the never before investigated Hedyosmum sprucei Solms (Chloranthaceae) essential oil (EO). The antioxidant activity, performed through DPPH bioautographic assay and spectrophotometric evaluations (IC50 = 230 ± 10 g/mL), seemed to be mainly due to - cadinol and -muurolol. (HP)TLC bioautography, focused on antimicrobial capacities, pointed out -cadinol, -muurolol, - muurolol, caryophyllene oxide, and methyleugenol as the most effective compounds against Staphylococcus aureus, considered as testing strain. Moreover, the microdilution method, assessed among a wide panel of microorganisms, revealed Listeria grayi and Staphylococcus aureus as the most sensitive among human tested strains and Clavibacter michiganensis among phytopathogens. GCMS chemical profile showed that bioactive molecules represented only a small quantity of the whole EO: germacrene D (23.16%), -caryophyllene (15.53%), -cadinene (5.50%), -copaene (5.08%), and -phellandrene (3.48%) were the main compounds, highlighting an uncommon composition among the genus Hedyosmum. Finally, H. sprucei EO was checked for cytotoxic potential against A549 (lung cancer) and MCF-7 (breast cancer) cell lines showing promising cytotoxic effects against both cell lines after 48 h (IC50 A549 = 44.05 ± 2.35 g/mL; IC50 MCF-7 = 32.76 ± 4.92 g/mL) and 72 h (IC50 A549 = 43.55 ± 2.80 g/mL; IC50 MCF-7 = 33.64 ± 0.43 g/mL

Biological Activities of Zingiber officinale Roscoe Essential Oil against Fusarium spp.: A Minireview of a Promising Tool for Biocontrol

Zingiber officinale Roscoe is an herbal plant native to Asia that can be found in all tropical countries. It is used in folk medicine, food, and cosmetics. A chemical characterization and some agronomic experiments have been carried out on Z. officinale essential oil, showing promising findings for the biological control of fungal pathogens belonging to the genus Fusarium. The aim of this review is to collect and update the literature covering its phytochemistry and biological activities as a Fusarium spp. plant-based biocide. The present research was conducted using the following bibliographic databases: Scifinder, Pubmed, and Science Direct. Thirteen papers were selected based on the adopted criteria. Data were independently extracted by the three authors of this work, and the final article selections were completed in a manner that avoided the duplication of data. The main chemical compounds were &alpha;-zingiberene, geranial, and aryl-curcumene, but a remarkable difference was found concerning the chemical compositions. Z. officinale essential oil was shown to possess promising biological functions against Fusarium spp. These findings offer new research approaches and potential applications as a biocontrol ingredient for Z. officinale essential oil