RESUMO
Novel approaches to the characterization of medicinal plants as biofactories have lately increased in the field of biotechnology. In this work, a multifaceted approach based on plant tissue culture, metabolomics, and machine learning was applied to decipher and further characterize the biosynthesis of phenolic compounds by eliciting cell suspension cultures from medicinal plants belonging to the Bryophyllum subgenus. The application of untargeted metabolomics provided a total of 460 phenolic compounds. The biosynthesis of 164 of them was significantly modulated by elicitation. The application of neurofuzzy logic as a machine learning tool allowed for deciphering the critical factors involved in the response to elicitation, predicting their influence and interactions on plant cell growth and the biosynthesis of several polyphenols subfamilies. The results indicate that salicylic acid plays a definitive genotype-dependent role in the elicitation of Bryophyllum cell cultures, while methyl jasmonate was revealed as a secondary factor. The knowledge provided by this approach opens a wide perspective on the research of medicinal plants and facilitates their biotechnological exploitation as biofactories in the food, cosmetic and pharmaceutical fields.
RESUMO
Phenolic compounds constitute an important family of natural bioactive compounds responsible for the medicinal properties attributed to Bryophyllum plants (genus Kalanchoe, Crassulaceae), but their production by these medicinal plants has not been characterized to date. In this work, a combinatorial approach including plant tissue culture, untargeted metabolomics, and machine learning is proposed to unravel the critical factors behind the biosynthesis of phenolic compounds in these species. The untargeted metabolomics revealed 485 annotated compounds that were produced by three Bryophyllum species cultured in vitro in a genotype and organ-dependent manner. Neurofuzzy logic (NFL) predictive models assessed the significant influence of genotypes and organs and identified the key nutrients from culture media formulations involved in phenolic compound biosynthesis. Sulfate played a critical role in tyrosol and lignan biosynthesis, copper in phenolic acid biosynthesis, calcium in stilbene biosynthesis, and magnesium in flavanol biosynthesis. Flavonol and anthocyanin biosynthesis was not significantly affected by mineral components. As a result, a predictive biosynthetic model for all the Bryophyllum genotypes was proposed. The combination of untargeted metabolomics with machine learning provided a robust approach to achieve the phytochemical characterization of the previously unexplored species belonging to the Bryophyllum subgenus, facilitating their biotechnological exploitation as a promising source of bioactive compounds.
RESUMO
The subgenus Bryophyllum includes about 25 plant species native to Madagascar, and is widely used in traditional medicine worldwide. Different formulations from Bryophyllum have been employed for the treatment of several ailments, including infections, gynecological disorders, and chronic diseases, such as diabetes, neurological and neoplastic diseases. Two major families of secondary metabolites have been reported as responsible for these bioactivities: phenolic compounds and bufadienolides. These compounds are found in limited amounts in plants because they are biosynthesized in response to different biotic and abiotic stresses. Therefore, novel approaches should be undertaken with the aim of achieving the phytochemical valorization of Bryophyllum sp., allowing a sustainable production that prevents from a massive exploitation of wild plant resources. This review focuses on the study of phytoconstituents reported on Bryophyllum sp.; the application of plant tissue culture methodology as a reliable tool for the valorization of bioactive compounds; and the application of machine learning technology to model and optimize the full phytochemical potential of Bryophyllum sp. As a result, Bryophyllum species can be considered as a promising source of plant bioactive compounds, with enormous antioxidant and anticancer potential, which could be used for their large-scale biotechnological exploitation in cosmetic, food, and pharmaceutical industries.
RESUMO
Plant nutrition is a crucial factor that is usually underestimated when designing plant in vitro culture protocols of unexploited plants. As a complex multifactorial process, the study of nutritional imbalances requires the use of time-consuming experimental designs and appropriate statistical and multiple regression analysis for the determination of critical parameters, whose results may be difficult to interpret when the number of variables is large. The use of machine learning (ML) supposes a cutting-edge approach to investigate multifactorial processes, with the aim of detecting non-linear relationships and critical factors affecting a determined response and their concealed interactions. Thus, in this work we applied artificial neural networks coupled to fuzzy logic, known as neurofuzzy logic, to determine the critical factors affecting the mineral nutrition of medicinal plants belonging to Bryophyllum subgenus cultured in vitro. The application of neurofuzzy logic algorithms facilitate the interpretation of the results, as the technology is able to generate useful and understandable "IF-THEN" rules, that provide information about the factor(s) involved in a certain response. In this sense, ammonium, sulfate, molybdenum, copper and sodium were the most important nutrients that explain the variation in the in vitro culture establishment of the medicinal plants in a species-dependent manner. Thus, our results indicate that Bryophyllum spp. display a fine-tuning regulation of mineral nutrition, that was reported for the first time under in vitro conditions. Overall, neurofuzzy model was able to predict and identify masked interactions among such factors, providing a source of knowledge (helpful information) from the experimental data (non-informative per se), in order to make the exploitation and valorization of medicinal plants with high phytochemical potential easier.
RESUMO
Organogenesis constitutes the biological feature driving plant in vitro regeneration, in which the role of plant hormones is crucial. The use of machine learning (ML) technology stands out as a novel approach to characterize the combined role of two phytohormones, the auxin indoleacetic acid (IAA) and the cytokinin 6-benzylaminopurine (BAP), on the in vitro organogenesis of unexploited medicinal plants from the Bryophyllum subgenus. The predictive model generated by neurofuzzy logic, a combination of artificial neural networks (ANNs) and fuzzy logic algorithms, was able to reveal the critical factors affecting such multifactorial process over the experimental dataset collected. The rules obtained along with the model allowed to decipher that BAP had a pleiotropic effect on the Bryophyllum spp., as it caused different organogenetic responses depending on its concentration and the genotype, including direct and indirect shoot organogenesis and callus formation. On the contrary, IAA showed an inhibiting role, restricted to indirect shoot regeneration. In this work, neurofuzzy logic emerged as a cutting-edge method to characterize the mechanism of action of two phytohormones, leading to the optimization of plant tissue culture protocols with high large-scale biotechnological applicability.
Assuntos
Aprendizado de Máquina , Organogênese , Reguladores de Crescimento de Plantas/farmacologia , Plantas Medicinais/crescimento & desenvolvimento , Compostos de Benzil/farmacologia , Ácidos Indolacéticos/farmacologia , Kalanchoe/efeitos dos fármacos , Kalanchoe/crescimento & desenvolvimento , Organogênese/efeitos dos fármacos , Plantas Medicinais/efeitos dos fármacos , Purinas/farmacologiaRESUMO
We combined machine learning and plant in vitro culture methodologies as a novel approach for unraveling the phytochemical potential of unexploited medicinal plants. In order to induce phenolic compound biosynthesis, the in vitro culture of three different species of Bryophyllum under nutritional stress was established. To optimize phenolic extraction, four solvents with different MeOH proportions were used, and total phenolic content (TPC), flavonoid content (FC) and radical-scavenging activity (RSA) were determined. All results were subjected to data modeling with the application of artificial neural networks to provide insight into the significant factors that influence such multifactorial processes. Our findings suggest that aerial parts accumulate a higher proportion of phenolic compounds and flavonoids in comparison to roots. TPC was increased under ammonium concentrations below 15 mM, and their extraction was maximum when using solvents with intermediate methanol proportions (55-85%). The same behavior was reported for RSA, and, conversely, FC was independent of culture media composition, and their extraction was enhanced using solvents with high methanol proportions (>85%). These findings confer a wide perspective about the relationship between abiotic stress and secondary metabolism and could serve as the starting point for the optimization of bioactive compound production at a biotechnological scale.
RESUMO
Numerous studies have been focused on hydrophobic compounds encapsulation as oils. In fact, oils can provide numerous health benefits as synergic ingredient combined with other hydrophobic active ingredients. However, stable microparticles for pharmaceutical purposes are difficult to achieve when commonly techniques are used. In this work, sunflower oil was encapsulated in calcium-alginate capsules by prilling technique in co-axial configuration. Core-shell beads were produced by inverse gelation directly at the nozzle using a w/o emulsion containing aqueous calcium chloride solution in sunflower oil pumped through the inner nozzle while an aqueous alginate solution, coming out from the annular nozzle, produced the beads shell. To optimize process parameters artificial intelligence tools were proposed to optimize the numerous prilling process variables. Homogeneous and spherical microcapsules with narrow size distribution and a thin alginate shell were obtained when the parameters as w/o constituents, polymer concentrations, flow rates and frequency of vibration were optimized by two commercial software, FormRules® and INForm®, which implement neurofuzzy logic and Artificial Neural Networks together with genetic algorithms, respectively. This technique constitutes an innovative approach for hydrophobic compounds microencapsulation.