From Ethnomedicine to Plant Biotechnology and Machine Learning: The Valorization of the Medicinal Plant Bryophyllum sp.

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 industriesThis research was funded by Xunta de Galicia through “Red de Uso Sostenible de los Recursos Naturales y Agroalimentarios” (REDUSO, grant number ED431D 2017/18) and “Cluster of Agricultural Research and Development” (CITACA Strategic Partnership, gran numbered ED431E 2018/07). The authors acknowledge the FPU grant awarded to Pascual García-Pérez from the Spanish Ministry of Education (grant number FPU15/04849)S

Machine Learning Technology Reveals the Concealed Interactions of Phytohormones on Medicinal Plant In Vitro Organogenesis

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 applicabilityThe authors acknowledge the FPU grant awarded to Pascual García-Pérez from the Spanish Ministry of Education (grant number FPU15/04849)S

Plant Antioxidants in Food Emulsions

Addition of free radical scavenging antioxidants (AOs) is one of practical strategies controlling the oxidative stability in food emulsions. Attention has been directed toward AOs derived from natural plant extracts with the capacity to improve health and well-being due to lack of consumers’ trust toward synthetic antioxidant in food. Nevertheless, antioxidant efficiency varies widely from one compound to another and the most abundant AOs in our diet are not necessarily those that have the best availability profile at the reaction place with free radicals. In this book chapter, we will provide a state-of-the-art summary of the uses of plant AOs in colloidal systems, ranging from their main structural features to their benefits for the human health and their antioxidant role in controlling the oxidative stress and, particularly, the oxidation of lipid-based food emulsions

Neurofuzzy logic predicts a fine-tuning metabolic reprogramming on elicited Bryophyllum PCSCs guided by salicylic acid

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.Xunta de Galicia | Ref. ED431E 2018/07Xunta de Galicia | Ref. ED431D2017/1

Plant antioxidants in food emulsions

Addition of free radical scavenging antioxidants (AOs) is one of practical strategies controlling the oxidative stability in food emulsions. Attention has been directed toward AOs derived from natural plant extracts with the capacity to improve health and well-being due to lack of consumers’ trust toward synthetic antioxidant in food. Nevertheless, antioxidant efficiency varies widely from one compound to another and the most abundant AOs in our diet are not necessarily those that have the best availability profile at the reaction place with free radicals. In this book chapter, we will provide a state-of-the-art summary of the uses of plant AOs in colloidal systems, ranging from their main structural features to their benefits for the human health and their antioxidant role in controlling the oxidative stress and, particularly, the oxidation of lipid-based food emulsions.Xunta de Galicia | Ref. ED431D-2017/18Ministerio de Educación y Ciencia | Ref. CTQ2006-13969-BQUMinisterio de Educación, Cultura y Deporte | Ref. FPU15/04849Xunta de Galicia | Ref. POS-B/2016/01

Phenolic profiling and in vitro bioactivities of three medicinal Bryophyllum plants

Bryophyllum constitutes a subgenus within the genus Kalanchoe that contains several plant species used in traditional medicine worldwide for the treatment of several diseases. However, little is known about the phytoconstituents of Bryophyllum spp. and previous reports have pointed at their low in Planta concentrations of bioactive compounds. In this work, we take advantage of plant in vitro culture for the study of the phenolic compounds found in the aerial parts of Bryophyllum spp. and their associated bioactivities. Our results show that the induction of nutritional stress leads to an improved accumulation of phenolic compounds, mainly flavonols and anthocyanins, represented by myricetin and malvidin glycosides, respectively. This effect is mainly found for B. × houghtonii, whose hydroethanolic extracts promoted the highest antioxidant, cytotoxic and anti-inflammatory activities. In the case of cytotoxic activity, Bryophyllum extracts showed an enhanced activity against the MCF-7 human breast carcinoma cell line. Meanwhile, extracts from B. daigremontianum promoted a wide range of effectiveness against different bacterial and fungal strains. This study is committed to shed light about the phytochemical potential associated to this unexplored subgenus, with the aim of considering Bryophyllum spp. as a valuable source of bioactive compounds for their exploitation in food, cosmetic and pharmaceutical industries.The authors acknowledge the Spanish Ministry of Education for the FPU grant awarded to Pascual García-Pérez (FPU15/04849) and ADICAM research center for kindly providing the plant material.info:eu-repo/semantics/publishedVersio

Modelling of the in vitro culture of Bryophyllum sp. for efficient biosynthesis of phenolic compounds with pharmacological potential

La planta de Kalanchoe es un endemismo propio de Madagascar, que se encuentra extendido a lo largo de África Oriental, Sudáfrica, Arabia y el Sudeste Asiático (Sharker et al., 2012). Se trata de un género taxonómicamente complejo, perteneciente a la familia de las Crasuláceas que comprende más de 125 especies (Gehrig et al., 2001). Debido a la creciente demanda de productos fitoterapeúticos para el tratamiento de enfermedades y, teniendo en cuenta, que estos compuestos bioactivos se sintetizan en concentraciones muy bajas en las plantas (p. ej.: para la obtención de tan sólo un gramo de bufadienólidos serían necesarias 5,5 t de raíz (Moniuszko-Szajwaj et al., 2016)); el cultivo in vitro se perfila como una estrategia prometedora para la producción a gran escala de compuestos bioactivos con propiedades farmacológicas. En este sentido, el cultivo in vitro de plantas, órganos y células de Bryophyllum, aportaría un mayor rendimiento en la producción de compuestos bioactivos, que el cultivo tradicional. Además, soslayaría sus inconvenientes, tales como su dependencia geoclimática, el cambio climático, la aparición de nuevas plagas agrícolas, la necesidad de grandes extensiones de tierra de cultivo, enorme gasto de agua o fluctuaciones en la producción interanual. En cambio, una de las grandes ventajas del cultivo in vitro, es que, además de ser independiente de todos esos condicionamientos climáticos y de sostenibilidad en los recursos (se desarrolla en condiciones controladas de cultivo), permite incrementar la producción de los compuestos bioactivos generando estrés (mediante elicitores) para inducir la respuesta defensiva de la planta que estimula su síntesis y su acumulación. Objetivos y contenidos básicos del trabajo: El objetivo principal de la tesis doctoral sería optimizar los medios de cultivo in vitro (planta, órganos y suspensiones celulares) de tres especies de Bryophyllum que se emplean en la medicina tradicional: B. daigremontianum, B. tubiflorum y la híbrida entre ambas, Bryophyllum × houghtonii. El segundo objetivo sería la identificación de nuevos compuestos bioactivos seleccionados de diversos órganos y de las suspensiones celulares, y la selección de aquellos de mayor interés farmacológico. Por último, diseñar un protocolo para maximizar la producción de compuestos bioactivos en suspensiones celulares, a partir de determinados órganos de la planta, en función de los resultados anteriores. Metodología: Hasta el momento y según nuestro conocimiento, apenas existe bibliografía sobre el cultivo in vitro de Bryophyllum y no existen publicaciones acerca de la obtención de suspensiones celulares, por tanto, se recurrirá a bibliografía de especies lo más próximas taxonómicamente posible, por lo que se llevará a cabo una extensa búsqueda bibliográfica. A continuación, comenzará la parte experimental con el establecimiento del cultivo in vitro de estas tres especies, diseñando medios de cultivo que maximicen la producción de cada una de ellas. Posteriormente, se identificarán los compuestos de interés y el órgano de la planta donde se acumulan preferentemente. Se aplicarán herramientas de inteligencia artificial (IA) para la modelización del cultivo in vitro. Las redes neuronales artificiales (ANN) y la lógica difusa se han empleado con éxito en la biotecnología vegetal. La modelización de procesos posibilita no solo la comprensión del proceso en sí, sino que también permite predecir y optimizar la producción de metabolitos (Gallego et al., 2011). Se utilizarán diversas técnicas avanzadas como HPLC-Masas, y “omics” (por ejemplo, la metabolómica) para el análisis, identificación y cuantificación.A planta de Kalanchoe é un endemismo propio de Madagascar, que se atopa estendido ao longo de África Oriental, Sudáfrica, Arabia e o Sueste Asiático ( Sharker et al., 2012). Trátase dun xénero taxonómicamente complexo, pertencente á familia das Crasuláceas que comprende máis de 125 especies ( Gehrig et al., 2001). Debido á crecente demanda de produtos fitoterapeúticos para o tratamento de enfermidades e, tendo en conta, que estes compostos bioactivos sintetízanse en concentracións moi baixas nas plantas (p. ex.: para a obtención de tan só un gramo de bufadienólidos serían necesarias 5,5 t de raíz ( Moniuszko- Szajwaj et al., 2016)); o cultivo in vitro perfílase como unha estratexia prometedora para a produción a gran escala de compostos bioactivos con propiedades farmacolóxicas. Neste sentido, o cultivo in vitro de plantas, órganos e células de Bryophyllum, achegaría un maior rendemento na produción de compostos bioactivos, que o cultivo tradicional. Ademais, eludiría os seus inconvenientes, tales como a súa dependencia geoclimática, o cambio climático, a aparición de novas pragas agrícolas, a necesidade de grandes extensións de terra de cultivo, enorme gasto de auga ou fluctuacións na produción interanual. Unha das grandes vantaxes do cultivo in vitro, é que, ademais de ser independente de todos eses condicionamentos climáticos e de sustentabilidade nos recursos (desenvólvese en condicións controladas de cultivo), permite incrementar a produción dos compostos bioactivos xerando estrés (mediante elicitores) para inducir a resposta defensiva da planta que estimula a súa síntese e a súa acumulación. Obxectivos e contidos básicos do traballo: O obxectivo principal da tese doutoral sería optimizar os medios de cultivo in vitro (planta, órganos e suspensións celulares) de tres especies de Bryophyllum que se empregan na medicina tradicional: B. daigremontianum, B. tubiflorum e a híbrida entre ambas, Bryophyllum × houghtonii. O segundo obxectivo sería a identificación de novos compostos bioactivos seleccionados de diversos órganos e das suspensións celulares, e a selección daqueles de maior interese farmacolóxico. Por último, deseñar un protocolo para maximizar a produción de compostos bioactivos en suspensións celulares, a partir de determinados órganos da planta, en función dos resultados anteriores. Metodoloxía: Ata o momento e segundo o noso coñecemento, apenas existe bibliografía sobre o cultivo in vitro de Bryophyllum e non existen publicacións acerca da obtención de suspensións celulares, por tanto, recorrerase a bibliografía de especies o máis próximas taxonómicamente posible, polo que levará a cabo unha extensa procura bibliográfica. A continuación, comezará a parte experimental co establecemento do cultivo in vitro destas tres especies, deseñando medios de cultivo que maximicen a produción de cada unha delas. Posteriormente, identificaranse os compostos de interese e o órgano da planta onde se acumulan preferentemente. Aplicaranse ferramentas de intelixencia artificial ( IA) para a modelización do cultivo in vitro. As redes neuronais artificiais (ANN) e a lóxica difusa empregáronse con éxito na biotecnoloxía vexetal. A modelización de procesos posibilita non só a comprensión do proceso en si, permitindo predicir e optimizar a produción de metabolitos (Gallego et al., 2011). Utilizaranse diversas técnicas avanzadas como HPLC-Masas, e “ omics” (por exemplo, a metabolómica) para a análise, identificación e cuantificación da produción dos compostos de interese en biorreactores a partir de suspensións celulares.The Kalanchoe plant is a endemism typical of Madagascar, which is spread throughout East Africa, South Africa, Arabia and Southeast Asia (Sharker et al., 2012). It is a taxonomically complex genus, belonging to the family of the Crasuláceas that includes more than 125 species (Gehrig et al., 2001). Due to the growing demand for phytotherapeutic products for the treatment of diseases and, taking into account, that these bioactive compounds are synthesized in very low concentrations in plants (e.g., to obtain only one gram of bufadienolides would be necessary 5.5 t of root (Moniuszko-Szajwaj et al., 2016)); In vitro culture is emerging as a promising strategy for large-scale production of bioactive compounds with pharmacological properties. In this sense, the in vitro culture of plants, organs and cells of Bryophyllum, would provide a greater yield in the production of bioactive compounds, than the traditional culture. In addition, it would avoid its inconveniences, such as its geoclimatic dependence, climate change, the appearance of new agricultural pests, the need for large areas of farmland, huge water expenditure or fluctuations in interannual production. On the other hand, one of the great advantages of in vitro culture is that, in addition to being independent of all these climatic conditions and sustainability of resources (it develops under controlled cultivation conditions), it allows to increase the production of bioactive compounds generating stress (by elicitors) to induce the defensive response of the plant that stimulates its synthesis and accumulation. Objectives and basic contents of the work: The main objective of the doctoral thesis would be to optimize the in vitro culture media (plant, organs and cell suspensions) of three species of Bryophyllum that are used in traditional medicine: B. daigremontianum, B. tubiflorum and the hybrid between them, Bryophyllum × houghtonii. The second objective would be the identification of new bioactive compounds selected from various organs and cell suspensions, and the selection of those of greater pharmacological interest. Finally, to design a protocol to maximize the production of bioactive compounds in cell suspensions, from certain organs of the plant, based on the previous results. Methodology: According to our knowledge, there is hardly any literature on the in vitro culture of Bryophyllum and there are no publications about obtaining cell suspensions, therefore, we will resort to bibliography of species as taxonomically as possible, so it will take conducted an extensive bibliographic search. Next, the experimental part will begin with the establishment of the in vitro culture of these three species, designing culture media that maximize the production of each of them. Subsequently, the compounds of interest and the organ of the plant where they accumulate preferentially will be identified. Artificial intelligence (AI) tools will be applied for in vitro culture modeling. Artificial neural networks (ANN) and diffuse logic have been used successfully in plant biotechnology. The modeling of processes allows not only the understanding of the process itself but also allows to predict and optimize the production of metabolites (Gallego et al., 2011). Various advanced techniques such as HPLC-Masses, and "omics" (for example, metabolomics) will be used for the analysis, identification and quantification of the production of the compounds of interest in bioreactors from cell suspensions

From ethnomedicine to plant biotechnology and machine learning: the valorization of the medicinal plant Bryophyllum sp.

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.Xunta de Galicia | Ref. ED431D 2017/18Xunta de Galicia | Ref. ED431E 2018/07Ministerio de Educación y Formación Profesional | Ref. FPU15 / 0484

Machine learning technology reveals the concealed interactions of phytohormones on medicinal plant In vitro organogenesis

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.Xunta de Galicia | Ref. REDUSO, ED431D 2017/18Clúster de Investigación y Desarrollo Agropecuario | Ref. CITACA, ED431E 2018/0

Machine learning unmasked nutritional imbalances on the medicinal plant Bryophyllum sp. cultured in vitro

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.Ministerio de Educación, Cultura y Deporte | Ref. FPU15/0484