The broad spectrum of bioactive properties of phenolic extracts: a prospective study in three different plants

Mestrado com dupla diplomação com o High Institute of Biotechnology of Monastir (ISBM), TunisiaNatural resources like plants are currently used all over developed and under developed countries of the world as traditional home remedies and are promising agents for drug discovery as they play crucial role in traditional medicine. The use of plants for medicinal purpose usually varies from country to country and region to region because their use depends on the history, culture, philosophy and personal attitudes of the users (Ahmad et al., 2015). The use of plants and plant products as drugs predates the written human history (Hayta et al., 2014). Plants are a very important resource for traditional drugs and around 80% of the population of the planet use plants for the treatment of many diseases and traditional herbal medicine accounts for 30-50% of the total medicinal consumption in China. In North America, Europe and other well-developed regions over 50% of the population have used traditional preparations at least once (Dos Santos Reinaldo et al., 2015). Medicinal plants have been used over years for multiple purposes, and have increasingly attract the interest of researchers in order to evaluate their contribution to health maintenance and disease’s prevention (Murray, 2004). Recently between 50,000 and 70,000 species of plants are known and are being used in the development of modern drugs. Plants were the main therapeutic agents used by humans from the 19th century, and their role in medicine is always topical (Hayta et al., 2014). The studies of medicinal plants are rapidly increasing due to the search for new active molecules, and to improve the production of plants or bioactive molecules for the pharmaceutical industries (Rates, 2001). Several studies have been reported, but numerous active compounds directly responsible for the observed bioactive properties remain unknown, while in other cases the mechanism of action is not fully understood. According to the WHO 25% of all modern medicines including both western and traditional medicine have been extracted from plants, while 75% of new drugs against infective diseases that have arrived between 1981 and 2002 originated from natural sources, it was reported that the world market for herbal medicines stood at over US $60 billion per year and is growing steadily (Bedoya et al., 2009). Traditional medicine has an important economic impact in the 21st century as it is used worldwide, taking advantage on the low cost, accessibility, flexibility and diversity of medicinal plants (Balunas & Kinghorn, 2005).Medicinal plants have received great attention in the last years because of their richness in bioactive compounds responsible for different biological activities. In the present study, hydroethanolic extracts of Hypericum androseamum L., Equisetum giganteum L. and Tilia platyphyllos Scop., were chemically characterized in terms of phenolic composition and evaluated for their antioxidant, antitumor and anti-inflammatory properties. All three medicinal plants displayed antioxidant activity as free radical scavengers but mainly as lipid peroxidation inhibitors. The extracts were also effective in inhibiting nitric oxide production, as an indicator of the anti-inflammatory potential, displayed antitumor properties in breast carcinoma (MCF-7), non-small cell lung cancer (NCI-H460), cervical carcinoma (HeLa) and hepatocellular carcinoma (HepG2) cell lines and, up to the maximal tested concentration, did not show any cytotoxic effects for non-tumor cells. 5-O-Caffeoylquinic acid and 3-O-caffeoylquinic acid were the most abundant phenolic compounds identified in the H. androsaemum extract, and might be responsible for the observed bioactivity. Protocatechuic acid and epicatechin were most abundant in the T. platyphyllos extract, while kaempferol-O-glucoside-O-rutinoside was the most abundant phenolic compound in the E. giganteum extract. The results showed that these medicinal plants displayed very interesting bioactivities, which are related with phenolic composition and quantity. However, it would be interesting to evaluate the in vivo efficacy of these plant extracts, as also their mechanisms of action, in order to establish real therapeutic doses

Hibiscus sabdariffa L. como uma fonte de nutrientes, compostos bioativos e agentes corantes

As plantas são uma fonte de pigmentos naturais que podem ser utilizados como corantes alimentares com baixo risco de toxicidade e com benefícios nutricionais e bioativos. Esta temática despertou o interesse da comunidade científica e desencadeou estudos com o objetivo de explorar matrizes naturais como fonte de compostos bioativos e corantes. Neste trabalho, a composição química de Hibiscus sabdariffa L. em compostos hidrofílicos (açúcares e ácidos orgânicos) e lipofílicos (ácidos gordos e tocoferóis) foi determinada por técnicas cromatográficas. Além disso, foram avaliadas as propriedades bioativas (antioxidante e antimicrobiana) e o perfil fenólico dos seus extratos aquoso e hidroetanólico. A frutose e a glucose foram os açúcares livres detetados, estando este último presente em maior quantidade (6,5±0,6 g/100 g massa seca). Foram identificados quatro ácidos orgânicos, ácido oxálico, málico, shiquímico e fumárico, destacando o ácido málico que foi encontrado em maior concentração (9,10±0,09 g/100 g). Quanto aos tocoferóis foram identificados os vitâmeros α e β-tocoferóis, sendo o α-tocoferol o mais abundante (39,19±0,06 mg/100 g). Na determinação do perfil de ácidos gordos, foram identificados 18 compostos, evidenciando-se o ácido linoleico com uma contribuição de 32,65±0,07%. Na avaliação do potencial antioxidante e antimicrobiano, ambos os extratos apresentaram atividade. Analisando o perfil fenólico, foram identificados 12 compostos, sendo o 5- (hidroximetil)furfural e a delfinidina-3-O-sambobiósido as moléculas não-antociânica e antociânica mais abundante, respetivamente. Tendo em conta os resultados obtidos, esta espécie revelou ser uma fonte promissora de compostos bioativos e corantes, que poderá ser explorada pelas indústrias alimentar, cosmética e farmacêutica.info:eu-repo/semantics/publishedVersio

Hibiscus sabdariffa L. as a source of nutrients, bioactive compounds and colouring agents

The nutritional and bioactive composition of plants have aroused much interest not only among scientists, but also in people's daily lives. Apart from the health benefits, plants are a source of pigments that can be used as natural food colorants. In this work, the nutritional composition of Hibiscus sabdariffa L. was analysed, as well as its bioactive compounds and natural pigments. Glucose (sugar), malic acid (organic acid), a-tocopherol (tocopherol) and linoleic acid (fatty acid) were the major constituents in the corresponding classes. 5-(Hydroxymethyl) furfural was the most abundant non-anthocyanin compound, while delphinidin-3-O-sambubioside was the major anthocyanin both in its hydroethanolic extract and infusion. H. sabdariffa extracts showed antioxidant and antimicrobial activities, highlighting that the hydroethanol extract presents not only lipid peroxidation inhibition capacity, but also bactericidal/fungicidal inhibition ability for all the bacteria and fungi tested. Furthermore, both extracts revealed the absence of toxicity using porcine primary liver cells. The studied plant species was thus not only interesting for nutritional purposes but also for bioactive and colouring applications in food, cosmetic and pharmaceutical industries.The authors 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) and under Programme NORTE2020 for the project NORTE-01-0145-FEDER-023289, as also for L. Barros contract. The authors are also grateful to the Interreg España-Portugal for financial support through the project 0377_Iberphenol_6_E.info:eu-repo/semantics/publishedVersio

Optimization of heat- and ultrasound-assisted extraction of anthocyanins from Hibiscus sabdariffa calyces for natural food colorants

calyces. The extraction variables, time (t), ethanol proportion (S), and temperature (T) or ultrasonic power (P), were combined in a 5-level experimental design and analysed by response surface methodology for process optimization. The delphinidin-3-O-sambubioside (C1) and cyanidin-3-O-sambubioside (C2) levels were monitored by LC-DAD-ESI/MSn and used as response criteria. The developed models were successfully fitted to the experimental data and used to determine optimal extraction conditions. UAE was the most efficient method yielding 51.76 mg C1+C2/g R under optimal conditions (t=26.1 min, P=296.6W and S=39.1% ethanol, v/ v). The dose-response effects of the solid/liquid ratio on the extraction rate were also determined. The anthocyanin levels herein reported are higher than those found in the literature, which support the potential use of H. sabdariffa as a sustainable source of natural colorants with application in different industrial sectors.The authors 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), FEDER through POCI-COMPETE2020 and FCT for financial support to LA LSRE-LCM (POCI-01-0145-FEDER-006984), J. Pinela (UID/AGR/00690/ 2013_DNAABN) and L. Barros contract. This work is funded by the European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope of Project NORTE- 01-0145-FEDER-023289: DeCodE and project Mobilizador Norte-01- 0247-FEDER-024479: ValorNatural®. The authors are also grateful to FEDER-Interreg España-Portugal programme for financial support through the project 0377_Iberphenol_6_E. To the Xunta de Galicia for financial support to M.A. Prieto.info:eu-repo/semantics/publishedVersio

Exploring the chemical and bioactive properties of: Hibiscus sabdariffa L. calyces from Guinea-Bissau (West Africa)

Different external factors influence the chemical characteristics of natural products, varying according to the geographic origin. The present study determined the nutritional and chemical composition of Hibiscus sabdariffa L. dried calyces (Guinea-Bissauan origin), as well as the phenolic composition, antioxidant, anti-inflammatory, cytotoxicity and antimicrobial activity of its infusion and hydroethanolic extracts. Among the chemical composition, glucose and quinic acid showed the major concentration for sugars and organic acids, respectively. Palmitic acid and α-tocopherol were the most abundant lipophilic compounds. The individual phenolic compounds were analysed through HPLC-DAD-ESI/MS. Thirteen compounds were identified in the hydroethanolic extract, while twelve were identified in the infusion, being 3- O-caffeoylquinic acid the major non-anthocyanin compound. Three anthocyanins were identified, being delphinidin-3-O-sambubioside the most abundant. Both extracts showed promising results in all the bioactive assays. This study exhibited beneficial properties of H. sabdariffa and also emphasized the potential applications of this plant in different industrial sectors.The authors 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), as also for L. Barros contract. The authors are also grateful to the Interreg España-Portugal for financial support through the project 0377_Iberphenol_6_E; European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope of Project NORTE-01-0145-FEDER-023289: DeCodE and project Mobilizador Norte-01-0247- FEDER-024479: ValorNatural®, and to the Ministry of Education, Sciences and Technological Development of Republic of Serbia, grant No. 173032.info:eu-repo/semantics/publishedVersio

Contribution of the phenolic composition to the antioxidant, anti-inflammatory and antitumor potential of Equisetum giganteum L. and Tilia platyphyllos Scop.

Naturally-occurring phytochemicals have received pivotal attention in the last few years, due to the increasing evidence of biological activities. Thus, in the present study, the antioxidant, anti-inflammatory and antitumor potentials of hydroethanolic extracts rich in phenolic compounds obtained from Equisetum giganteum L. and Tilia platyphyllos Scop. were assessed and directly correlated with their content of phenolic compounds, by using HPLC-DAD-ESI/MS analysis. T. platyphyllos showed the higher bioactive potential, evaluated in terms of antioxidant (radical scavenging effects-105 µg mL -1 , reducing power-123 µg mL -1 , ß-carotene bleaching inhibition-167 µg mL -1 , and lipid peroxidation inhibition-56 µg mL -1 ), anti-inflammatory (225 µg mL -1 inhibited 50% of nitric oxide production) and antitumor (breast-224 µg mL -1 ; lung-247 µg mL -1 ; cervical-195 µg mL -1 and hepatocellular-173 µg mL -1 carcinoma cells) activity, without having cytotoxic effects ( > 400 µg mL -1 ). These biological properties were positively correlated with its content and composition of phenolic compounds. Flavonoid contents were markedly higher than the content of phenolic acids, in both samples, being respectively 50.4 mg g -1 and 11.65 mg g -1 for T. platyphyllos, and 21.7 mg g -1 and 4.98 mg g -1 for E. giganteum. Moreover, while in E. giganteum extract, kaempferol-O-glucoside-O-rutinoside was the most abundant flavonoid, in T. platyphyllos extract protocatechuic acid and (-)-epicatechin were the most abundant phenolic acid and flavonoid, respectively. In relation to their content of phenolic acids, protocatechuic and caffeic acids existed in higher abundance in T. platyphyllos and E. giganteum hydroethanolic extracts, respectively. However, it would be interesting to evaluate the in vivo efficacy of both plant extracts to unveil the involved modes of action and to establish effective therapeutic doses.The authors are grateful to Fundação para a Ciência e a Tecnologia (FCT, Portugal) for financial support to CIMO (Pest-OE/AGR/UI0690/2015) and to POCI-01-0145-FEDER- 006984 (LA LSRE-LCM) funded by ERDF through POCICOMPETE2020 and FCT. L. Barros and N. Martins thank FCT for their grants (SFRH/BPD/107855/2015 and SFRH/BD/87658/ 2012, respectively). The GIP-USAL is financially supported by the Spanish Government through the project AGL2015-64522- C2-2-R.info:eu-repo/semantics/publishedVersio

HPLC-DAD-ESI/MS phenolic characterization and biological activity of Equisetum giganteum L.

Naturally-occurring phytochemicals have received a pivotal attention in the last years, due to the increasing evidences of biological activities. Equisetum giganteum L., commonly known as “giant horsetail”, is a native plant from Central and South America, being largely used in dietary supplements as diuretic, hemostatic, antiinflammatory and anti-rheumatic agents [1,2]. The aim of the present study was to evaluate the antioxidant (scavenging effects on 2,2-diphenyl-1-picrylhydrazyl radicals- RSA, reducing power- RP, β-carotene bleaching inhibition- CBI and lipid peroxidation inhibition- LPI), anti-inflammatory (inhibition of NO production in lipopolysaccharidestimulated RAW 264.7 macrophages) and cytotoxic (in a panel of four human tumor cell lines: MCF-7- breast adenocarcinoma, NCI-H460- non-small cell lung cancer, HeLa- cervical carcinoma and HepG2- hepatocellular carcinoma; and in non-tumor porcine liver primary cells- PLP2) properties of E. giganteum, providing a phytochemical characterization of its extract (ethanol/water, 80:20, v/v), by using highperformance liquid chromatography coupled to diode array detection and electrospray ionisation mass spectrometry (HPLC-DAD–ESI/MS). E. giganteum presented fourteen phenolic compounds, two phenolic acids and twelve flavonol glycoside derivatives, mainly kaempferol derivatives, accounting to 81% of the total phenolic content, being kaempferol-O-glucoside-O-rutinoside, the most abundant molecule (7.6 mg/g extract). The extract exhibited antioxidant (EC50 values = 123, 136, 202 and 57.4 μg/mL for RSA, RP, CBI and LPI, respectively), anti-inflammatory (EC50 value = 239 μg/mL) and cytotoxic (GI50 values = 250, 258, 268 and 239 μg/mL for MCF-7, NCI-H460, HeLa and HepG2, respectively) properties, which were positively correlated with its concentration in phenolic compounds. Furthermore, up to 400 μg/mL, it did not revealed toxicity in non-tumor liver cells. Thus, this study highlights the potential of E. giganteum extracts as rich sources of phenolic compounds that can be used in the food, pharmaceutical and cosmetic fields

Caracterização química e bioatividades de Hibiscus sabdariffa L.

A grande diversidade na composição química das plantas é responsável pelas suas múltiplas aplicações. Para além das propriedades bioativas, as plantas são também fonte de nutrientes e metabolitos secundários nomeadamente, pigmentos naturais que podem ser utilizados em alternativa aos corantes artificiais [1,2]. Neste trabalho, foi determinada a composição de Hibiscus sabdariffa L. em açúcares, ácidos gordos, ácidos orgânicos e tocoferóis, tendo sido também avaliado o potencial bioativo e a composição em fenólicos dos seus extratos aquoso e hidroetanólico. O perfil individual de açúcares foi determinado por HPLC-RI, os ácidos gordos por GC-FID, os ácidos orgânicos por HPLC-DAD e os tocoferóis por HPLC-fluorescência. A análise de compostos fenólicos foi efetuada por HPLC-DAD-ESI/ MS, enquanto que as propriedades bioativas foram avaliadas através de ensaios de atividade antioxidante e antimicrobiana. A hepatotoxicidade dos extratos foi também testada através de uma cultura primária de células de fígado de porco. A glucose (açúcar), o ácido málico (ácido orgânico), o α- tocoferol (tocoferol) e o ácido linoleico (ácido gordo) foram os principais constituintes nas classes correspondentes. 5-(Hidroximetil)furfural foi o composto não-antociânico mais abundante, enquanto delfinidina-3-O-sambobiósido foi a antocianina presente em maior concentração em ambos os extratos testados. Apesar de ambos os extratos de H. sabdariffa terem demonstrado atividade antioxidante e antimicrobiana, destaca-se o extrato hidroetanólico com uma maior capacidade de inibição da peroxidação lipídica em homogeneizados de células cerebrais de porco e maiores efeitos bactericidas e fungicidas. Foi também evidente que nenhum dos extratos analisados revelou hepatotoxicidade. Deste modo, a espécie H. sabdariffa revelou ser interessante não só como fonte de nutrientes, mas também de compostos bioativas e pigmentos, com enorme interesse para as indústrias alimentar, cosmética e farmacêutica.À Fundação para a Ciência e a Tecnologia (FCT, Portugal) e ao FEDER, no âmbito do programa PT2020, pelo apoio financeiro ao CIMO (UID/AGR/00690/2013) e ao contrato de L. Barros. Ao programa Interreg España-Portugal pelo apoio financeiro através do projeto 0377_Iberphenol_6_E.info:eu-repo/semantics/publishedVersio

Optimization and comparison of maceration and ultrassound assisted extraction systems for the production of a colorant extract from Hibiscus sabdariffa L. flowers

Hibiscus sabdariffa L. is an herbaceous medicinal plantused as infusion for the treatment of several disorders, namely, fever and high blood pressure, gastrointestinal and hepatic diseases. In addition to those health benefits, the calyces area potential source of natural pigments, due to its high content in anthocyanins. This work aimed to optimize the experimental conditions of anthocyanins’extraction from dried calyces of H. sabdariffa, comparing two methodologies, maceration and ultrasound assisted extraction, in order to develop a natural colorant. To obtain the conditions that maximize anthocyanins’ extraction, a response surface methodology (RSM) was applied using the circumscribed central composite design of three variables with five levels. Time, temperature and ethanol - water proportion - in the case of maceration, were the relevant independent variables, while for ultrasound assisted extraction, the ultrasonic power was used instead of temperature which was kept constant. Two anthocyanin compounds were identified by HPLC-DAD-ESI/MS: delphinidin3-sambubioside and cyanidin-3-sambubioside. The responses used as criteria were the quantification of the identified anthocyanins and the extraction yield of the final residue. Ultrasound assisted extraction proved to be the most effective method: 43 min, 390 W and 46 % of ethanol. At these optimal conditions, the extraction yield was 61.2%, with a total anthocyanins’ content of 23.1 mg/g dried plant, and 47.5 mg/g extract. Additionally, the response values were slightly improved by studying the solid/liquid ratio effect using the optimal conditions in dose-response format (5 to 200 g/L), showing continuously decreasing values as the solid/liquid ratio increases. Overall, the obtained results showed the possibility of using H. sabdariffa flowers as a source of anthocyanin compounds for industrial applications.European Union Regional Development Fund: Programme Portugal-NORTE2020 (Project NORTE-01-0145-FEDER-023289: DeCodE; Project NORTE-01-0247-FEDER- 024479: ValorNatural®); Programmme INTERREG V-A Espanha-Portugal (POCTEP) 2014- 2020 (Project 0377_Iberphenol_6_E). And Xunta de Galicia, Spain, for M.A. Prieto grant.info:eu-repo/semantics/publishedVersio

Hibiscus sabdariffa L. calyces as a source of anthocyanins rich extracts to be used as a natural food colouring agente

Colour is the most important sensory perception when it comes to setting peoples expectations regarding the likely taste and flavour of food [l]. Since the consumers are more aware regarding the additives that are incorporated into food products, they have shown a preference for the consumption of natural additives over the artificial ones. This hás been one ofthe research lines ofthe scientific community, searching for natural additives to replace the artificial counterparts [2]. The present work studies the stability of an extract obtained from hibiscus calyces (Hibiscus sabdariffa L.), to test its possible use as a natural colourant in foods. The aqueous stability of the extract was monitored using the total anthocyanins content (TAC) as response (determined by HPLC-DAD) in function of time (t, 14 days), temperature (T, 4 to 70 °C), and pH (2 to 6). Aided by mechanistic/phenomenological models, the conditions favouring the stabilization ofthe extract were provided. The results obtained showed that the aqueous stability of the H. sabdariffa extracts was higher at lower pH values, shorter t values and T between 4 to 35 °C. However, certain new stable combinations of conditions were found, providing a wide range of conditions, in which the TAC ofH. sabdariffa extracts will remain stable at values of T within the range of50 to 70°C. Such facts, will help to understand how to incorporate stable natural extracts, rich in TAC, as natural colorant additives in food products, that may require strong processing conditions, such as bakery and dairy products. These results provide useful information for: i) potential industrial use offí. sabdariffa extract as alternative sources of natural additives rich in TAC; and ii) shelf-life calculations and TAC loss predictions at specific conditions of T and pH. Finally, the results obtained showed a certain agreement with previous reports regarding the stability of anthocyanin compounds, but providing a new alternative source.The authors are grateful to the Poundation tor Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financiai support to CIMO (UID/AGR/00690/2013) and L. Barras contract; European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope ofProject NORTE-01-0145-FEDER-023289 DeCodE and Mobilizing Project Norte-01-0247-FEDER-024479 ValorNatural*.info:eu-repo/semantics/publishedVersio