Development of cosmeceutical formulations based on mushroom extracts and their individual compounds

[ES]La industria cosmética está constantemente en busca de ingredientes de origen natural debido a sus propiedades multifuncionales, su eficacia competitiva, y su menor toxicidad. Por otra parte, los hongos han sido una parte importante de nuestra dieta durante años debido a su riqueza nutricional y su abundante contenido en biomoléculas; sin embargo, su potencial cosmecéutico y nutricosmético no ha sido plenamente explotado. Este estudio tuvo como objetivo la optimización, utilizando la metodología de superficie de respuesta (RSM), de la extracción de compuestos bioactivos a partir de Ganoderma lucidum y Agaricus blazei y la comparación de técnicas de extracción convencionales con métodos más sostenibles. Se llevaron a cabo estudios adicionales relacionados con su composición química y valor nutricional (por ejemplo, azúcares, ácidos orgánicos, tocoferoles, y ácidos grasos). Además, extractos obtenidos, utilizando el sistema de extracción de Soxhlet, a partir de esos hongos junto con otros procedentes de Agaricus bisporus, Pleurotus ostreatus y extractos de Lentinula edodes, se analizaron en términos de actividad anti-inflamatoria, anti-tirosinasa, antioxidante, y antimicrobiana seguido de su caracterización en términos de ácidos fenólicos y compuestos relacionados, ergosterol, y triterpenoides. Además, los extractos obtenidos fueron incorporados en una crema cosmética base para evaluar propiedades bioactivas de soporte. Además, para evaluar la contribución individual como cosmecéuticos del ácido p-hidroxibenzoico, ácido pcumárico, ácido protocatéquico, ácido cinámico y ergosterol, fueron estudiadas sus propiedades anti-inflamatorias, anti-tirosinasa, y actividades antimicrobianas, seguido de la incorporación de estos compuestos en una crema cosmética base. Debido a la posibilidad de que los extractos y los compuestos individuales puedan perder su potencia con el tiempo, ya sea debido a la oxidación o la degradación, se prepararon en forma de micropartículas usando la técnica de atomización/coagulación. Las micropartículas se caracterizaron en términos de morfología, distribución de tamaño de partícula, y la eficiencia de encapsulación. A continuación, dichas micropartículas se incorporaron en una crema cosmético de base y se comparó su comportamiento frente a las formas libres (formulaciones que contienen extractos libres/compuestos individuales). Los extractos más prometedores y sus correspondientes formulaciones fueron sometidos a test de evaluación in vitro de su seguridad utilizando los ensayos de MTT y LDH en líneas celulares de queratinocitos humanos (HaCaT) y fibroblastos (HFF-1). Además, los extractos fueron sometidos a estudios ex vivo de permeabilidad de la piel utilizando una celda de Franz provista con una membrana natural obtenida a partir de la piel de oreja de cerdo para determinar la biodisponibilidad tópica de los compuestos bioactivos presentes en los extractos. El estudio de optimización en la recuperación de compuestos bioactivos de Ganoderma lucidum y A. blazei mediante RSM mostró que los valores predichos por los modelos estaban muy de acuerdo con las observaciones experimentales. Además, se concluyó que el rendimiento de extracción de compuestos bioactivos a partir de los hongos estudiados era altamente dependiente de las variables de extracción (tiempo, temperatura, proporción de disolvente, la relación sólido-líquido, y potencia). A. blazei y G. ludidum también mostraron un elevado valor nutricional. Se cuantificaron a-tocoferol, ácido oxálico y veintiún ácidos grasos (principalmente PUFA) en A. blazei. G. lucidum, por su parte, mostró un perfil de ácidos grasos similares (PUFA˃SFA˃MUFA) y con la presencia de α-tocoferol y δ tocoferol. Comparativamente, A. blazei reveló un menor contenido de grasa, pero mayor aporte de energía. Se observó que el efecto sobre la viabilidad celular del extracto preparado a partir de A. blazei en líneas celulares Caco-2 y HT29-MTX se mantenía de una manera concentracióndependiente. Los extractos etanólicos obtenidos a partir de los hongos estudiados mostraron actividad antioxidante, anti-inflamatoria, anti-tirosinasa y antimicrobiana, y después de la incorporación en la crema cosmético base, las formulaciones cosmecéuticas finales conservaron algunas de estas bioactividades. El extracto de G. lucidum se encontró que era una fuente rica de triterpenoides debido principalmente a la alta contribución de ácidos ganodéricos A, C2, y H, mientras que los ácidos fenólicos (ácido p-hidroxibenzoico, p-cumárico, ácido protocatéquico y ácido cinámico) y ergosterol se identificaron en A. bisporus, P. ostreatus, L. edodes y extractos de A. blazei. Cada uno de estos compuestos también mostraron efectos antiinflamatorios, anti-tirosinasa y actividades antimicrobianas. Además, se utilizaron con éxito micropartículas para encapsular los extractos/compuestos individuales con alta eficiencia de encapsulación y rendimiento. Después de la incorporación en la crema cosmética base de los extractos/compuestos individuales libres y las micropartículas producidas a partir de ellos, las formas encapsuladas permitieron una liberación controlada que conduce a una mayor preservación de la bioactividad cuando se compara con las formas libres. Debido a que presentaban el potencial cosmecéutico más interesante, los extractos de P. ostreatus y G. lucidum fueron sometidos a estudios toxicológicos, que revelaron ausencia de toxicidad en líneas celulares de queratinocitos y fibroblastos, lo cual es indicativo de su potencial seguridad para ser utilizados como ingredientes cosmecéuticos. Los ácidos protocatequico y siríngico fueron los únicos compuestos que permeaban a partir del extracto de G. lucidum, mientras que se observó una muy baja permeación para los compuestos presentes en el extracto de P. ostreatus. También se obtuvieron resultados similares para la formulación preparada usando ambos extractos. Los resultados mostraron que los triterpenoides presentes en G. lucidum podrían ser potenciadores de la permeabilidad natural, lo que corrobora la idoneidad del uso de estos extractos de hongos como ingredientes para el cuidado de la piel. En general, los resultados mostraron que los productos para el cuidado de la piel se pueden formular usando extractos de hongos y que estos extractos tienen el potencial de ser utilizados a gran escala. Por lo tanto, la integración de los residuos generados por la industria de transformación de hongos, que pueden servir como materia prima para la obtención de extractos bioactivos, puede intensificarse.[EN]The cosmetic industry is constantly in search of ingredients from natural sources because of their multifunctional properties, competitive effectiveness, and lower toxicity effects. Otherwise, mushrooms have been an important part of our diet for years due to their rich nutritional and abundant content in biomolecules; however, their cosmeceutical and nutricosmetic potential have not been fully exploited. This study aimed at optimizing the extraction of bioactive compounds from Ganoderma lucidum and Agaricus blazei by Response Surface Methodology (RSM) and comparing conventional extraction techniques with more sustainable methods. Further studies regarding their nutritional and chemical composition (e.g., sugars, organic acids, tocopherols, and fatty acids), were conducted. Also, extracts obtained from those mushrooms, but also from Agaricus bisporus, Pleurotus ostreatus and Lentinula edodes extracts, using the Soxhlet extraction system, were analysed in terms of antiinflammatory, anti-tyrosinase, antioxidant, and antimicrobial activities followed by their characterization in terms of phenolic acids and related compounds, ergosterol, and triterpenoids. Furthermore, the obtained extracts were incorporated in a base cosmetic cream to ascertain bioactive properties sustainment. Moreover, to evaluate the individual cosmeceutical contribution of p-hydroxybenzoic acid, p-coumaric acid, protocatechuic acid, cinnamic acid and ergosterol, their anti-inflammatory, anti-tyrosinase, and antimicrobial activities were studied, followed by incorporation of these compounds in a base cosmetic cream. Because of the possibility of the extracts and the individual compounds to lose their potency over time either due to oxidation or degradation, microparticles were prepared using the atomization/coagulation technique. The microparticles were characterized in terms of morphology, particle size distribution, and encapsulation efficiency. This is followed by incorporation of the produced microparticles into a base cosmetic cream and comparison of their performance with the free forms (formulations containing free extracts/individual compounds). The most promising extracts and their corresponding formulations were submitted to in vitro safety evaluation using the MTT and LDH assays in keratinocyte (HaCaT) and fibroblast (HFF-1) cell lines. Moreover, they were submitted to ex vivo skin permeation studies using a Franz diffusion apparatus with pig ear skin as permeation membrane to determine topical bioavailability of the bioactive compounds present in the extracts. The optimization study on bioactive compounds recovery from G. lucidum and A. blazei using RSM showed that the values predicted by the models were in close agreement with experimental observations. Furthermore, it was concluded that the extraction yield of bioactive compounds from the studied mushrooms was highly dependent on the extraction variables (time, temperature, solvent proportion, solid-liquid ratio, and power). A. blazei and G. ludidum also showed high nutritional contribution. α-Tocopherol, oxalic acid and twenty-one fatty acids (mainly PUFA) were quantified in A. blazei. G. lucidum showed a similar fatty acids’ profile (PUFA˃SFA˃MUFA) and with the presence of α-tocopherol and δ-tocopherol. Comparatively, A. blazei revealed lower fat content, but higher energy contribution. The cell viability effect of the extract prepared from A. blazei on Caco-2 and HT29 MTX cells was found to be maintained in a concentration-dependent manner. The ethanolic extracts obtained from the studied mushrooms showed antioxidant, antiinflammatory, anti-tyrosinase and antimicrobial activities, and after incorporation into the base cosmetic cream, the final cosmeceutical formulations preserved some of these bioactivities. G. lucidum extract was found to be a rich source of triterpenoids mainly due to the high contribution of ganoderic acid A, C2, and H, while phenolic acids (phydroxybenzoic acid, pcoumaric acid and protocatechuic acid), the related compound cinnamic acid, and ergosterol were identified in A. bisporus, P. ostreatus, L. edodes and A. blazei extracts. Each one of these compounds also showed anti-inflammatory, anti-tyrosinase and antimicrobial activities. Furthermore, microparticles were successfully used to encapsulate extract/individual compounds with high encapsulation efficiency and yield. After incorporation of the free extracts/individual compounds and the produced microparticles into the base cosmetic cream, the encapsulated forms allowed for a controlled release leading to bioactivity preservation when compared with the free forms. Because they presented the most interesting cosmeceutical potential, P. ostreatus and G. lucidum extracts were submitted to toxicological studies, revealing no toxicity in keratinocytes and fibroblast cell lines, which is indicative of their potential safety to be used as cosmeceutical ingredients. Protocatechuic acid and syringic acid were the only compounds permeating from the G. lucidum extract, while very low detection was observed for compounds present in the P. ostreatus extract. Similar results were also obtained for the formulation prepared using both extracts. The results showed that tritepenoids in G. lucidum might be natural permeation enhancers and these results pointed out for the suitability of using these mushroom extracts as skin care ingredients. Overall, the results showed that skin care products can be formulated using mushroom extracts and that these extracts have the potential to be utilised at large scale use. Hence, integration of wastes generated by the mushroom processing industry, which can serve as raw materials for the obtainment of bioactive extracts, can be intensified

Anti-inflammatory potential of mushroom extracts and isolated metabolites

Background: In the recent years natural resources are being in focus due to their great potential to be exploited in the discovery/development of novel bioactive compounds and, among them, mushrooms can be highlighted as alternative sources of anti-inflammatory agents. Scope and approach: The present review reports the anti-inflammatory activity of mushroom extracts and of their bioactive metabolites involved in this bioactive action. Additionally the most common assays used to evaluate mushrooms anti-inflammatory activity were also reviewed, including in vitro studies in cell lines, as well as in animal models in vivo. Key findings and conclusions: The anti-inflammatory compounds identified in mushrooms include polysaccharides, terpenes, phenolic acids, steroids, fatty acids and other metabolites. Among them, polysaccharides, terpenoids and phenolic compounds seem to be the most important contributors to the anti-inflammatory activity of mushrooms as demonstrated by numerous studies. However, clinical trials need to be conducted in order to confirm the effectiveness of some of these mushroom compounds namely, inhibitors of NF-κB pathway and of cyclooxygenase related with the expression of many inflammatory mediators

Hypericum genus cosmeceutical application - a decade comprehensive review on its multifunctional biological properties

Hypericum is a genus of flowering plants and presents a nearly worldwide distribution. The relevance of its species is mainly based on the occurrence of specialised secondary metabolites exhibiting a wide range of pharmaceutical and cosmeceutical properties. The most well-reported species in the last decade is H. perforatum, together with its exceptional polycyclic polyprenylated acylphloroglucinols metabolites. Several inhibitory enzyme properties such as anti-tyrosinase, anti-hyaluronidase, anti-collagenase, and anti-elastase, along with the relevant cosmeceutical anti-inflammatory and antimicrobial activities, have been reported in numerous biological studies concerning this genus. From 2010 to date were published more than 3000 articles; therefore, an updated summary of Hypericum species and their related cosmeceutical bioactive compounds is necessary. This detailed analysis will be mainly focused on cosmeceutical-related bioactivity, phytochemical composition, and the products and processes that have been developed using the generated knowledge. Despite all the progress made in recent years, which contributed to a state-of-the-art improvement, only a few studies regarding the genus have been dedicated to investigating the mechanisms of action and the safety (toxicology) of its bioactive compounds using well-validated methodologies. A strategic partnership between RD and vital cosmeceutical enterprises could be the requisite to promote the development of a higher number of efficient and marketable Hypericum-derived cosmeceutical products.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020). L. Barros is also thankful to FCT through the institutional scientific employment program-for her contract. Ana Rita Silva is also grateful to FCT for her Doctoral Grant (SFRH/BD/145834/2019). Oludemi Taofiq is grateful to the European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope of Project Mobilizador Norte-01- 0247-FEDER-024479: ValorNatural® for his contract.info:eu-repo/semantics/publishedVersio

Mushrooms extracts and compounds in cosmetics, cosmeceuticals and nutricosmetics—a review

The cosmetic industry is constantly in search of ingredients from natural sources because of their competitive effectiveness and lower toxicity effects. Mushrooms have been an important part of our diet for years and are now finding their way as cosmetic ingredients, either as cosmeceutical or as nutricosmetics. The present review focuses on the most relevant activities of mushroom extracts, as well as on their bioactive compounds, which make them interesting ingredients for cosmetic formulations. Mushroom extracts, as well as their bioactive metabolites, revealed anti-tyrosinase, anti-hyaluronidase, anti-collagenase and anti-elastase activity. Emphasis was also given to their important anti-oxidant, antimicrobial and anti-inflammatory potential, topics largely studied by numerous authors, making them very versatile and multi-functional cosmetic ingredients. Some of the bioactive compounds and the mechanism responsible for the activities ascribed to mushrooms were highlighted. Other activities were identified as needing to be further studied in order to identify the major compounds contributing to the target activity, as well as their mechanisms of action. Based on the above findings, mushroom extracts, as well as their bioactive metabolites, constitute important ingredients that can help to combat aging, reduce the severity of inflammatory skin disease and correct hyperpigmentation disorders. These findings and claims must be correctly supported by clinical trials and in vivo studies.The authors acknowledge FCT (Portugal) for financial support to CIMO (PEst-OE/AGUI0690/2015) and to FCT/MEC and FEDER under Programme PT2020 for financial support to LSRE (Project UID/EQU/50020/2013), and to QREN, ON2 and FEDER (Projects NORTE-07-0124-FEDER-000014 and NORTE-07-0162-FEDER-000050)

Mushroom ethanolic extracts as cosmeceuticals ingredients: Safety and ex vivo skin permeation studies

Mushrooms are important sources of natural bioactive compounds that are increasingly used as cosmeceutical ingredients. In this context, ethanolic extracts were prepared from Ganoderma lucidum and Pleurotus ostreatus and tested by incorporation into base cosmetic creams. In vitro safety evaluation of the extracts and cosmetic formulations prepared therefore was carried out using the MTT and LDH assays in keratinocyte (HaCaT) and fibroblast (HFF-1) cell lines. Moreover, they were submitted to ex vivo skin permeation studies using a Franz diffusion apparatus with pig ear skin as permeation membrane. The results showed the absence of toxicity for keratinocytes and fibroblasts in a concentration dependent manner, which is indicative of the safety of these extracts for cosmeceutical ingredients purposes. Protocatechuic and syringic acids were the only compounds permeating from G. lucidum extract in the first 8 h of study, no penetration was observed for phenolic acids found in P. ostreatus extract and formulation. These results pointed out for the suitability of using mushroom extracts as skin care ingredients and may contribute for the valorisation of wastes generated by the mushroom processing industry, which can serve as raw-materials for the obtainment of the extracts.The authors are grateful to the Foundation for Science and Technology and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2019) and Associate Laboratory LSRE-LCM (UID/EQU/50020/2019) funded by national funds through FCT/ MCTES (PIDDAC). This work is funded by the European Agricultural Fund for Rural Development (EAFRD), through the Rural Development Program (PDR2020), within the scope of Project MicoCoating (PDR2020-101-031472). Francisca Rodrigues is thankful for her postdoc research grant from the project Operação NORTE-01-0145- FEDER- 000011. This work received financial support from the European Union (FEDER funds through COMPETE), under the Partnership Agreement PT 2020, and National Funds (FCT, Foundation for Science and Technology) through project LAQV/UID/QUI/50006/ 2013 and NORTE-07-0124-FEDER-000069 – Food Science.info:eu-repo/semantics/publishedVersio

Response surface methodology and artificial neural network modeling as predictive tools for phenolic compounds recovery from olive pomace

This study optimized the extraction of three major phenolic compounds (oleuropein, tyrosol, and verbascoside) from olive pomace using microwave- and ultrasonic-assisted methods. Screening factorial design (SFD) and central composite design (CCD) were employed, and response surface methodology (RSM) and artificial neural networks (ANN) were used for data modeling. The microwave-assisted method in the SFD yielded higher compound amounts, with verbascoside showing a four-fold increase compared to the ultrasonic-assisted method. Factors like vessel diameter, ultrasonic power using UAE, and solvent acidity in both techniques had minimally impacted extractability. CCD-RSM revealed temperaturés significantly affect on oleuropein, but improved tyrosol recovery, with the effect on verbascoside being influenced by the temperature range. RSM and ANN integration enhanced understanding and prediction of factor behavior. Microwave-assisted extraction at 113 ◦C for 26 min, with minimum ramp time of 7.7 min, yielded 67.4, 57, and 5.1 mg of oleuropein, tyrosol, and verbascoside per gram of extract, respectively, with a prediction error ranging from 0.83 to 15.19.The authors are grateful to the Foundation for Science and Technology (FCT) for financial support to CIMO (UIDB/00690/2020 and UIDP/00690/2020), SusTEC (LA/P/0007/2020), L. Barros institutional contract, and Ana Rita Silva Doctoral Grant (SFRH/BD/145834/2019). To the ERDF through the Regional Operational Program North 2020, within the scope of the project OliveBIOextract (NORTE-01-0247- FEDER-049865). B. Melgar thanks the ERDF through the Regional Operational Program North 2020 for his contract within the Project OleaChain (NORTE-06-3559-FSE-000188). To MICINN for supporting the JDC contract of T. Oludemi (FJC2019-042549-I). Manuel Ayuso thanks PRIMA and FEDER-Interreg Espana- Portugal programme for financial support through the Local-NutLeg project (Section 1 2020 Agrofood Value Chain topic 1.3.1info:eu-repo/semantics/publishedVersio

Agaricus blazei Murrill from Brazil: An ingredient for nutraceutical and cosmeceutical applications

Operations for sorting mushrooms at the industrial level usually generate large amounts of bio-residues not conforming to strict morphological criteria for commercial purposes, even though their biological content is not compromised. In this context, the present work aimed at evaluating the potential for reutilizing industrially discarded Agaricus blazei Murill (ABM). Thus, the content of essential nutrients and the chemical composition were determined, and MTT and LDH assays were used to evaluate the viability and cell death of Caco-2 and HT29 cell lines of an ethanolic extract prepared from ABM (preliminary safety tests for nutraceutical applications). The extract was incorporated into a semi-solid base cosmetic cream and cell viability effects of the extract, and of the final cream formulation, on a keratinocyte cell line (HaCaT) were studied (preliminary safety tests for cosmeceutical applications). Essential nutrients, such as proteins and carbohydrates, and a low fat content were determined for ABM. Twenty-two fatty acids were detected, with polyunsaturated fatty acids (PUFA) (∼53%) being the most abundant fraction. The cell viabilities of Caco-2 and HT29 cells were maintained up to 100 μg mL−1. After incorporation into the base cream, a formulation with a pale yellow colour and favourable pH was obtained. The cell viability of HaCaT cells in the presence of the extract and the final cream formulation was maintained in a concentration dependent manner, which indicates the safety of this extract for cosmeceutical applications. The results suggest that ABM residues can be used as an inexpensive and sustainable source of nutraceutical and cosmeceutical ingredients.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and the FEDER under Programme PT2020 for providing financial support to CIMO (UID/AGR/ 00690/2013). This work was funded by the European Agricultural Fund for Rural Development (EAFRD) through the Rural Development Program (PDR2020) within the scope of Project MicoCoating (PDR2020-101-031472), which also supported Oludemi Taofiq’s research grant. Francisca Rodrigues is thankful to the project Operação NORTE-01-0145-FEDER-000011 for her postdoc research grant. Rosane Marina Peralta is a recipient of project number 307944/2015-8 from the CNPq (Conselho Nacional de Pesquisa e Desenvolvimento). This work received financial support from the European Union (FEDER funds through COMPETE) under Partnership Agreement PT2020 and National Funds (FCT, Foundation for Science and Technology) through project LAQV/UID/QUI/50006/2013 and NORTE-07-0124- FEDER-000069 – Food Science. This work also received financial support through the project POCI-01-0145 FEDER-006984 (LA LSRE-LCM), funded by FEDER, through POCI-COMPETE2020 and FCT.info:eu-repo/semantics/publishedVersio

Extraction of triterpenoids and phenolic compounds from: Ganoderma lucidum: optimization study using the response surface methodology

Chitosan is widely used in the preparation of organic-inorganic composite materials, such as n-HAp/CS composites, which find application for bone regeneration. The methods for their preparation are various, and usually based on the preparation of intermediate n-HAp/CS dispersions, which can greatly influence the final properties of the resulting composites since it is expected that homogenous and stable dispersions lead to composite materials with improved final properties. This work hypothesizes that, additionally to process parameters such as pH, n-HAp/CS weight ratio, mixing conditions and the presence of salts, chitosan itself has a high impact on dispersions stability. Thus, the importance of properly control the preparation of the n-HAp/CS intermediate dispersions is highlighted by doing a systematic study where relevant processing parameters were studied at lab scale using ultrasonication, alone or in the presence of chitosan, namely on particle size and zeta potential. Furthermore, and based on the best laboratorial conditions, the production of n-HAp/CS nanocomposite dispersions in continuous mode was attempted through NETmix® technology, an innovative static mixer and reactor developed at the Associate Laboratory LSRE-LCM of the Faculty of Engineering of the University of Porto (FEUP).Financial support for this work was provided in part by project Project POCI-01-0145-FEDER-006984 – Associate Laboratory LSRE-LCM funded by FEDER through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) – and by national funds through FCT - Fundação para a Ciência e a Tecnologia , and by “ AIProcMat@N2020 - Advanced Industrial Processes and Materials for a Sustainable Northern Region of Portugal 2020 ”, with reference NORTE-01-0145-FEDER-000006, supported by NORTE 2020 under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) . FCT and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2013). G. Ruphuy thanks Universidad de Costa Rica (UCR) and Ministerio de Ciencia, Tecnología y Telecomunicaciones de Costa Rica (MICITT) for her scholarship. Authors thank Fluidinova S.A. for providing the HAp samples.info:eu-repo/semantics/publishedVersio

Optimization of the extraction of phenolic compounds from Ganoderma lucidum

Ganoderma lucidum (Curtis) P. Karst, a rich source of several bioactive compounds, has been utilized for its medicinal and nutraceutical purposes since earliest history. Among its bioactive constituents, the phenolic content has been widely reported to be responsible for some its bioactive properties. Recent trends in bioactive compounds’ recovery have been focused on finding the best techniques that maximally extract valuable compounds from natural matrices. This study is aimed at optimizing the extraction of phenolic compounds (Ph) from G. lucidum using conventional techniques such as Soxhlet extraction (SE), heat assisted extraction (HAE) and comparing with ultrasound assisted extraction (UAE), considered a more sustainable method. The SE technique was optimized by applying 7 cycles while to effectively carry out the optimization of HAE and UAE, response surface methodology (RSM) was applied using a circumscribed central composite design with three independent variables (time, ethanol content, temperature or ultrasonic power). The extraction yield and total Ph were maximized and the optimal conditions were determined. For the SE technique, a positive linear dependency was achieved for the extraction yield, while for the Ph content expressed in terms of mg equiv. gallic acid (EGA) per g dw (dry weight), a significantly increase was observed up to the 5th cycle before a gradual decrease. The conditions that maximize the responses (extraction yield and total phenolics) were: 78.9 min, 90.0 °C and 62.5 % ethanol and 40 min, 100.0 W and 89.5% ethanol for HAE and UAE, respectively. The latter was the most effective, capable of achieving 4.9 ± 0.6 % extraction yield and 106.6 ± 16.2 mg EGA/g dw. The extraction process optimization proved to be a viable option for obtaining phenolic rich extracts from G. lucidum that can be used as bio-based ingredients.info:eu-repo/semantics/publishedVersio

Bioactive properties of greenhouse-cultivated green beans (Phaseolus vulgaris L.) under biostimulants and water-stress effect

The scarcity of irrigation water is severely affecting global crop production. In this context, biostimulants are increasingly used as alternatives means against abiotic stress conditions. In this study, phenolic compounds composition and bioactive properties of common bean (Phaseolus vulgaris L.) plants grown under water stress conditions and biostimulants application were investigated. RESULTS: Sixteen individual phenolic compounds were detected in both pods and seeds with a notable difference in their compositional profile. A significant effect on phenolic compounds content and composition was also observed for the biostimulants tested. Regarding the antibacterial activity, pods of the second harvest and seed extracts showed significant efficacy against Bacillus cereus, especially in water-stressed plants, where all biostimulant treatments weremore effective than positive controls. Moreover, all biostimulant treatments for seed extracts of water-stressed plants were more effective against Staphylococcus aureus compared with ampicillin, whereas streptomycin showed the best results. Extracts from pods of the secondharvest fromnormally irrigatedplants showed thebest results against the fungi tested, except for Penicillium verrucosum var. cyclopium. Finally, no significant cytotoxic effects were detected. CONCLUSION: In conclusion, the biostimulants tested increased total phenolic compounds content compared with control treatment, especially in pods of the first harvest and seeds of water-stressed plants. Moreover, bioactive properties showed a varied response in regard to irrigation and biostimulant treatment. Therefore, biostimulants can be considered as a useful means towards increasing phenolic compoundscontent, and theymay also affect the antimicrobial properties of pods and seeds extracts.We are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2013), A. Fernandes and L. Barros contract. We are also grateful to the FEDER–Interreg España–Portugal Programme for financial support, through the project 0377_Iberphenol_6_E, and to the Ministry of Education, Science and Technological Development, grant no. 173032.info:eu-repo/semantics/publishedVersio