Caracterização do perfil fenólico por HPLC-DAD-ESI / MS de flores comestíveis das espécies I. balsamina e I. walleriana

Devido à exigência dos consumidores, a indústria alimentar tem procurado matrizes mais naturais para obtenção de ingredientes (Pires et., 2019). Com isso, as flores comestíveis têm despertado interesse por apresentarem agentes bioativos na sua composição (Fernandes et al., 2017). O género Impatiens é popularmente conhecido por suas flores de coloração atrativa, e possuem nos seus órgãos vegetais compostos capazes de proporcionar benefícios à saúde humana (Szewczyk., 2018).info:eu-repo/semantics/publishedVersio

Current status of genus Impatiens: Bioactive compounds and natural pigments with health benefits

The market for edible flowers has grown exponentially due to consumers demand. This has been motivated by the concern to include healthier foods in the daily diet, leading to a great interest in these natural matrices. However, and although the consumption of this type of matrices are popularly associated with health benefits, scientific studies are still scarce. The industry seeks to satisfy the consumers demands and, at the same time, seeks to launch new challenges in the sector, in order to outline new conservation and production strategies. Although there are several flowers with edible potential in nature, many are still little explored or unnoticed, so this type of study is increasingly sought after. Scope and approach: The present review intendeds to explore the context of edible flowers as well as their conditions of production and consumption. Then, focusing on the characterization of the genus Impatiens, which, although is still little explored, gathers data on its chemical and nutritional characterization, bioactivities, and the possible exploration of natural pigments and industrial application. Key findings and conclusions: Several studies have been focused on the chemical characterization of flowers, evaluation their edibility, as well as validating their bioactive potential. In particular, the genus Impatiens is characterized by presenting perennial and succulent herbs with attractive colors, normally found in tropical forests. There are countless studies on the bioactive activities present in these edible flowers. In addition, its pigmentation has a direct relationship with the presence of several compounds, especially anthocyanins and their derivatives. These bioactive compounds have a great interest to the food industry and consumers, due to their diverse health benefits that are provided by their regular consumption.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support by national funds FCT/ MCTES to CIMO (UIDB/00690/2020). L. Barros would like to thank the national funding by FCT, P.I., through the institutional scientific employment program-contract. To the project AllNat for the contract of C. Caleja (Project AllNat POCI-01-0145-FEDER-030463). The authors are also grateful to the project TRANSCoLAB 0612_TRANS_CO_LAB_2_P. This work is funded by 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 ®, Norte-01-0145-FEDER-000042: Project GreenHealth. This work was also financed by ERDF, through the Incentive System to Research and Technological Development, within the Portugal2020 Competitiveness and Internationalization Operational Program, for the project POCI-01-0247-FEDER-046112: BIOMA.info:eu-repo/semantics/publishedVersio

The compositional aspects of edible flowers as an emerging horticultural product

Edible flowers are becoming very popular, as consumers are seeking healthier and more attractive food products that can improve their diet aesthetics and diversify their dietary sources of micronutrients. The great variety of flowers that can be eaten is also associated with high variability in chemical composition, especially in bioactive compounds content that may significantly contribute to human health. The advanced analytical techniques allowed us to reveal the chemical composition of edible flowers and identify new compounds and effects that were not known until recently. Considering the numerous species of edible flowers, the present review aims to categorize the various species depending on their chemical composition and also to present the main groups of compounds that are usually present in the species that are most commonly used for culinary purposes. Moreover, special attention is given to those species that contain potentially toxic or poisonous compounds as their integration in human diets should be carefully considered. In conclusion, the present review provides useful information regarding the chemical composition and the main groups of chemical compounds that are present in the flowers of the most common species.info:eu-repo/semantics/publishedVersio

Chemical and bioactive characterization of Impatiens balsamina L. pink flowers and their application in a Portuguese pastry product

The rose petals of the species Impatiens balsamina L. were investigated. In this way the nutritional composition, phenolic profile and its bioactive composition were evaluated. Next, the extract obtained was applied to a product of the Portuguese pastry "bombocas". Proving to be a possible promising natural colour for the food industry.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); national funding by FCT, P.I., through the institutional and individual scientific employment program-contract for L. Barros, R. Calhelha, M.I. Dias and J. Pinela’s (CEECIND/01011/2018) contracts, respectively; research contract of C. Caleja (Project AllNatt, POCI- 01-0145-FEDER- 030463); and to FEDER-Interreg España-Portugal program for financial support through the project TRANSCoLAB 0612_TRANS_CO_LAB_2_P, BIOMA (POCI_01_0247_FEDER_046112) and Green Health (Norte- 01-0145-FEDER-000042. This work has been supported by the Ministry of Education, Science and Technological Development of Republic of Serbia (451-03-68/2020-14/200007).info:eu-repo/semantics/publishedVersio

Study on the potential application of Impatiens balsamina l. flowers extract as a natural colouring ingredient in a pastry product

Flowers of the genus Impatiens are classified as edible; however, their inclusion in the human diet is not yet a common practice. Its attractive colours have stirred great interest by the food industry. In this sense, rose (BP) and orange (BO) I. balsamina flowers were nutritionally studied, followed by an in-depth chemical study profile. The non-anthocyanin and anthocyanin profiles of extracts of both flower varieties were also determined by high-performance liquid chromatography coupled to a diode array and mass spectrometry detector (HPLC-DAD-ESI/MS). The results demonstrated that both varieties presented significant amounts of phenolic compounds, having identified nine non-anthocyanin compounds and 14 anthocyanin compounds. BP extract stood out in its bioactive properties (antioxidant and antimicrobial potential) and was selected for incorporation in “bombocas” filling. Its performance as a colouring ingredient was compared with the control formulations (white filling) and with E163 (anthocyanins) colorant. The incorporation of the natural ingredient did not cause changes in the chemical and nutritional composition of the product; and although the colour conferred was lighter than presented by the formulation with E163 (suggesting a more natural aspect), the higher antioxidant activity could meet the expectations of the current high-demand consumer.This research was funded by the European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope of Project GreenHealth—Digital strategies in biological assets to improve well-being and promote green health, Norte-01-0145-FEDER- 000042. This work has also been supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (451-03-68/2020-14/200007). The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support by national funds FCT/MCTES to CIMO (UIDB/00690/2020); national funding by F.C.T. and P.I., through the institutional scientific employment program-contract for M.I. Dias, C. Pereira, R.C. Calhelha, and L. Barros contracts, and through the individual scientific employment program-contract for M. Carocho contract (CEECIND/00831/2018) and to the Project Mobilizador Norte-01-0247-FEDER-024479: ValorNatural® for the contract of E. Pereira.info:eu-repo/semantics/publishedVersio

Chemical composition and bioactive characterisation of Impatiens walleriana

The attractive colour characteristics of the flowers of the species Impatiens walleriana have been arousing great interest in the food industry, which is looking for potential natural sources of colouring ingredients. In this sense, the present work focused on the chemical and bioactive characterization of pink and orange flowers of I. walleriana. The phenolic compounds were determined by HPLC-DAD-ESI/MS; in addition, different bioactivities (antioxidant, antimicrobial, anti-inflammatory and cytotoxicity) were also analysed. Both samples studied showed significant amounts of phenolic compounds, especially phenolic acids, flavonoids, and anthocyanins, which justifies the excellent performance in the different bioactivities studied. The orange variety, despite having a greater variety of phenolic compounds, showed a total amount of compounds lower than the pink variety. Overall, the flowers of I. walleriana emerge as a promising resource to be explored by the food industry.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support by national funds FCT/MCTES to CIMO (UIDB/00690/2020). M.I. Dias, R.C. Calhelha, C. Perreira, and L. Barros would like to thank the national funding by FCT, P.I., through the institutional scientific employment program-contract for their contracts; To the project AllNat for the contract of C. Caleja (Project AllNat POCI-01-0145-FEDER-030463) and to the Project Mobilizador Norte-01-0247-FEDER-024479: ValorNatural® for the contract of E. Pereira. The authors are also grateful to FEDER-Interreg España-Portugal programme for financial support through the project TRANSCoLAB 0612_TRANS_CO_LAB_2_P and to European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope of Project Mobilizador Norte-01-0247-FEDER-024479: ValorNatural® and Norte-01-0145-FEDER-000042: GreenHealth. This work has also been supported by the Ministry of Education, Science and Technological Development of Republic of Serbia (451-03-68/2020-14/200007).info:eu-repo/semantics/publishedVersio

Caracterização química e bioativa de diferentes flores do género Impatiens e estudo do seu potencial uso na indústria alimentar

As Plantas Alimentares Não Convencionas (PANCs) são plantas consideradas potencialmente adequadas para consumo humano, que geralmente crescem espontaneamente no ambiente sem condições prévias de cultivo e/ou tratamento. Apesar de muitas dessas plantas estarem disponíveis em abundância na natureza, são ainda pouco conhecidas pela população que as classifica como “ervas daninhas”. Dentro do universo das PANCs, as flores comestíveis destacam-se como uma fonte de nutrição utilizadas desde a antiguidade, normalmente classificadas como excelentes fontes de substâncias benéficas para a saúde humana. As flores do género Impatiens são muito utilizadas para fins ornamentais em projetos de paisagismo, e apesar de serem comestíveis, o seu uso em aplicações alimentares ainda não é prática comum. As cores atrativas características destas flores tem vindo a despertar grande interesse por parte da indústria alimentar que procura fontes naturais de ingredientes corantes. A associação de efeitos adversos com o consumo excessivo de alguns aditivos artificiais tendem a afetar as escolhas dos consumidores que atualmente, exigem produtos mais saudáveis e mais naturais. Desta forma, são várias as matrizes vegetais que tem vindo a ser exploradas. Neste sentido, este trabalho visou a identificação botânica e a caracterização colorimétrica das pétalas de duas espécies de Impatiens, I. balsamina e I. walleriana, nas variedades rosa e laranja de ambas. De um modo mais aprofundado, os compostos fenólicos foram determinados por cromatografia líquida de alta eficiência acoplada a detetor de díodos e detetor de espetrometria de massa (HPLC-DAD-ESI/MS) e foram analisadas diferentes bioatividades (atividades antioxidante, antimicrobiana, anti-inflamatória e citotoxicidade). Todas as amostras estudadas apresentaram quantidades expressivas de compostos fenólicos, em especial ácidos fenólicos e flavonoides. A espécie I. balsamina apresentou uma maior variedade de compostos fenólicos, no entanto, ambas as variedades rosa foram as detentoras da maior quantidade de compostos tanto antociânicos como não antociânicos. Esse resultado vai de encontro aos obtidos nas bioatividades, nos quais I. balsamina rosa se destacou como o extrato com potencial bioativo mais promissor. Ambas as variedades de I. balsamina foram caracterizadas química e nutricionalmente seguindo metodologias oficiais de análise AOAC. As flores apresentaram elevado teor em água (90%) e baixas quantidades de macronutrientes e cinzas. A variedade laranja apresentou teores mais elevados de gordura (0,12 ± 0,01 g/100 g pf) e proteínas (0,33 ± 0,01 g/100 g pf), enquanto a variedade rosa apresentou valores mais elevados de hidratos de carbono (4,76 ± 0,02 g/100 g pf) e valor energético (21,145 ± 0,003 kcal/100 g pf). Relativamente ao perfil em ácidos gordos, a variedade laranja apresentou teor mais elevado em ácidos gordos saturados (44,9 ± 0,5 %) enquanto que a variedade rosa apresentou predominância dos polinsaturados (52,7 ± 0,5 %), nos quais os ácidos esteárico (C18:0), linoleico (C18:2n6) e γ-linoleico (C18:3n6) foram os maioritários. Por sua vez, o ácido succínico surgiu como o ácido orgânico maioritário para ambas as variedades. Tendo em vista a possível aplicação industrial como ingrediente corante natural o extrato de pétalas rosa de Impatiens balsamina foi selecionado para incorporação em recheio de “bombocas”, comparando os resultados obtidos com “bombocas” tradicionais (recheio branco) e de recheio rosa (preparadas utilizando gelatina comercial de morango com corante E163). Notou-se que o extrato das flores de I. balsamina apresentou resultados notáveis, sendo capaz de proporcionar aumento razoável do teor proteico do produto final e realçar sua coloração. Apesar de a cor conferida pelo extrato ser um pouco mais clara que a apresentada pela gelatina de morango, esta parece sugerir um aspeto mais natural que, em adição com a atividade funcional conferida, atende às expetativas atuais dos consumidores, apresentando potencial de aplicação na indústria alimentar.Non-Conventional Food Plants (PANCs) are plants considered to be potentially suitable for human consumption, which generally grow spontaneously in the environment under previous conditions of cultivation and / or treatment. Although many of these plants are available in abundance in nature, they are still little known by the population that classifies them as "weeds". Within the universe of PANCs, edible flowers stand out as a source of nutrients used since ancient times, normally classified as excellent sources of substances beneficial to human health. Flowers of the genus Impatiens are widely used for ornamental purposes in landscaping projects, and although they are edible, their use in food applications is not yet common practice. The attractive colors characteristic of these flowers has aroused great interest on the part of the food industry that seeks natural sources of coloring ingredients. The association of adverse effects with the excessive consumption of some artificial additives has been affecting the choices of consumers who currently demand healthier and more natural products. In this way, several plant matrices have been explored. In this sense, this work aimed to characterize the petals of two species of Impatiens, I. balsamina and I. walleriana, in the pink and orange varieties of both. The phenolic compounds were determined by high performance liquid chromatography coupled to a diode detector and mass spectrometry detector (HPLC-DAD-ESI / MS) and different bioactivities (antioxidant, antimicrobial, anti-inflammatory and cytotoxicity activities) were analyzed. All samples studied showed significant amounts of phenolic compounds, especially phenolic acids and flavonoids. The species I. balsamina showed a greater variety of phenolic compounds; however, both pink varieties had the greatest amount of both anthocyanin and non-anthocyanin compounds. This result is in line with those obtained in bioactivities, in which I. balsamina pink stood out as the extract with the most promising bioactive potential. Both varieties of I. balsamina were chemically and nutritionally characterized following official AOAC analysis methodologies. The flowers had a high water content (90%) and low amounts of macronutrients and ash. The orange variety had higher levels of fat (0.12 ± 0.01 g / 100 g fw) and proteins (0.33 ± 0.01 g / 100 g fw), while for the pink variety it had higher values of carbohydrates (4.76 ± 0.02 g / 100 g fw) and energy value (21.145 ± 0.003 kcal / 100 g fw). Regarding the fatty acid profile, the orange variety had a higher content of saturated fatty acids (44.9 ± 0.5%) while the pink variety showed a predominance of polyunsaturated fatty acids(52.7 ± 0.5%), in which the stearic acids (C18:0), linoleic (C18:2n6) and γ-linoleic (C18:3n6) the majority. In turn, succinic acid emerged as the major organic acid for both varieties. Given the possible of the possible industrial application as natural coloring ingredients, the extract of I. balsamina rose petals was selected for incorporation into “bombocas” filling, comparing with traditional “bombocas” (white filling) and pink filling (prepared using commercial gelatine of strawberry with dye E163). The extract of the flowers of I. balsamina showed remarkable results, being able to provide a reasonable increase in the protein content of the final product and enhance its color. Although the color conferred by the extract is a little lighter than that presented by strawberry gelatin, it seems to suggest a more natural aspect that, in addition to the functional activity conferred, meets the current expectations of consumers, presenting potential for application in the food industry.Este trabalho é financiado pelo programa FEDER-Interreg España-Portugal, no âmbito do projeto 0377_Iberphenol_6_E e TRANSCoLAB 0612_TRANS_CO_LAB_2_P. Este trabalho foi financiado pelo Fundo Europeu de Desenvolvimento Regional (FEDER) através do Programa Operacional Regional Norte 2020, no âmbito do Projeto Mobilizador Norte-01-0247-FEDER-024479: ValorNatural®

Chemical characterization and bioactive properties of Impatiens Balsamina L. flowers

There is a growing interest from the scientific community in studying edible flowers in what concerns their possible application at an industrial level, which may be justified by the presence of several compounds with bioactive properties [1]. The genus Impatiens comprises approximately 500 species of perennial herbs, which stand out for their attractive coloring capacity [2]. Given the growing consumers’ awareness regarding the safety and beneficial properties of natural additives, the food industry has been motivated to explore natural colorant ingredients, producing healthier foodstuff [3]. In this sense, the present study intends to chemically and nutritionally characterize Impatiens balsamina L. flowers, as well as their bioactive properties in order to evaluate their potential application in the food industry. The nutritional profile (ash, protein, fat, and carbohydrate content and energy value) was evaluated using official analysis methods (AOAC). The phenolic compounds were analyzed by HPLC-DAD-ESI/MS in the ethanolic extracts. Furthermore, the ethanolic extracts were also evaluated for their antioxidant potential by the inhibition of oxidative hemolysis (OxHLIA) assay; their cytotoxic capacity in four human tumor cell lines by the sulforhodamine B method; and finally, their anti-inflammatory potential in macrophage rat cells. The flowers presented a high-water content (94.97±0.08%), as expected, and proteins (0.32±0.01 g/100g fw) stood out as the major macronutrients. Fructose and glucose were the only sugars identified, with a total of 1.34±0.01g/100g fw, being fructose the most abundant one. Regarding the phenolic composition, 9 non-anthocyanin and 7 anthocyanin compounds were tentatively identified. Kaempherol-O-acetylhexoside-hexoside and malvidin-O-acetylhexoside-Odeoxyhexoside- hexoside were the main non-anthocyanin and anthocyanin compounds found, respectively. Moreover, the ethanolic extract revealed anti-inflammatory activity and cytotoxic capacity for all the tested cell lines. In conclusion, this study showed satisfactory results regarding the presence of bioactive compounds in the ethanolic extracts of Impatiens flowers. Therefore, a more in-depth study of the optimal conditions for the extraction of high added-value coloring compounds would be of great interest to evaluate the real potential of this plant to be used by the food industry.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UIDB/00690/2020); national funding by FCT, P.I., through the institutional scientific employment program-contract for L. Barros, R. Calhelha, M.I. Dias and C. Pereira’s contract though the celebration of program-contract foreseen in No. 4, 5 and 6 of article 23º of Decree-Law No. 57/2016, of 29th August, amended by Law No. 57/2017, of 19th July; research contract of C. Caleja (Project AllNatt, POCI- 01-0145-FEDER-030463) and of E. Pereira (Project Mobilizador Norte-01-0247-FEDER-024479: ValorNatural®); and to FEDERInterreg España-Portugal programme for financial support through the projects 0377_Iberphenol_6_E, TRANSCoLAB 0612_TRANS_CO_LAB_2_P and Green Health (Norte-01-0145-FEDER-000042).info:eu-repo/semantics/publishedVersio

Tradescantia zebrina Bosse: study of the phenolic composition and bioactive properties of a potential natural coloring ingredient

Tradescantia zebrina Bosse is a succulent pantropical herb, native to Central America with a high capacity to grow in various conditions [1]. This plant is popularly used in cooking and traditional medicine [2]. Some species of the Transdecantia genus are described as possessing exceptionally stable anthocyanic compounds in plant organs, a condition of extreme importance for application in the food industry that is currently looking for potential natural coloring ingredients [3]. In this sense, the present work aimed to explore the phenolic composition and bioactive properties of the aerial parts T. zebrina extract. The phenolic composition of the extract was defined by High Performance Liquid Cromatography (HPLC-DAD-ESI/MS) and allowed the identification of 11 anthocyanins glycoside derivatives. Regarding the bioactive properties, i) the antioxidant activity was tested using an oxidative hemolysis assays (OxHLIA), reducing power, and free radical scavenging assay (DPPH); ii) cytotoxicity was evaluated in human tumor cells (MCF-7, breast carcinoma; NCI-H460, lung cancer; AGS, gastric carcinoma; and CaCo, colorectal adenocarcinoma) and hepatotoxicity in non-tumor cells (PLP2, pig liver; and Vero, monkey kidney cell) by the sulphorodamine B method; iii) the antimicrobial activity was evaluated against a panel of twelve food pathogens (S. aureus, B. cereus, L. monocytogenes, E. coli, S. Typhimurium, E. cloacae, A. fumigatus, A. niger, A. versicolor, P. funiculosum, P. verrucosum var. ciclopium and T. viride) using the microdilution method. The analyzed extract presented promising antioxidant activity, with promising EC50 values for all the testes assays, ranging from of 0.190 ± 0.001 mg/mL (DPPH), 0.061 ± 0.002 mg/mL (reducing power), and 0.057 ± 0.001 mg/mL (OxHILIA). Furthermore, the extract did not demonstrate toxicity at the maximum tested concentration (>400 μg/mL), but a remarkable activity against gastric carcinoma (AGS) was detected. Additionally, the extract showed excellent results in antibacterial and antifungal performance against all strains tested. In conclusion, it can be observed that the extract obtained from the aerial parts of T. zebrina represents themselves a natural antioxidants of great value for the industry, which, in addition to conferring colour to food products due to the quantity of anthocyanins, can contribute to the preservation of food 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), for E.O. PIRES JR. grant (2021.05425.BD), and for the contracts of C. Pereira, M.I. Dias, R.C. Calhelha, and L. Barros through the institutional scientific employment program-contract. To the project Healthy-PETFOOD for the contract of C. Caleja (Project Healthy-PETFOOD (POCI-01-0247-FEDER-047073) and to the Valor Natural project for the contract of E. Pereira (Mobilized Project Norte-01-0247-FEDER-024479). This work is funded by the European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope of Project GreenHealth - Digital strategies in biological assets to improve well-being and promote green health, Norte-01- 0145FEDER-000042 and by FEDER-Interreg España-Portugal programme for financial support through the project TRANSCoLAB 0612_TRANS_CO_LAB_2_P. This work has been supported by the Ministry of Education, Science and Technological Development of Republic of Serbia (451-03-68/2020-14/200007).info:eu-repo/semantics/publishedVersio

Bioactive profile of the extract of the petals of Impatiens walleriana as a natural food coloring alternative

In addition to improving sensory aspects, the incorporation of edible flowers in the human diet has been described as able to promote benefits for consumer health, mainly associated with their phenolic composition [1]. Impatiens walleriana Hook.f. is an edible ornamental plant, originally from East Africa and popularly recognized for its attractive colorful flowers [2]. The bioactivities present in flowers of the genus Impatiens have auspicious potential for the food, pharmaceutical and cosmetic industries [3]. In this sense, the present work focused on the chemical and bioactive characterization of flowers of Impatiens walleriana Hook species, particularly the pink and orange varieties. The phenolic compounds were determined by high performance liquid chromatography coupled to a diode detector and a mass spectrometry detector (HPLC-DAD-ESI/MS) and the bioactive compounds were evaluated through in vitro assays by determining the antioxidant (through the inhibition of the oxidative hemolysis assay, OxHLIA), antimicrobial (by means of a panel of six bacteria and six fungi) and anti-inflammatory activities (in mouse macrophage cells) and cytotoxicity (in tumor and non-tumor cell lines using the sulforhododamine B method). Both studied samples showed significant amounts of phenolic compounds, namely four phenolic acids, one flavonone and ten anthocyanins (divided into malvidins, pelargonidins and peonidins) being that the orange variety showed a total amount of compounds lower than the pink variety. Likewise, although both extracts presented a good bioactive performance in all tests carried out, the pink variety stood out as being superior compared to the orange variety. Overall, the flowers of I. walleriana emerge as a promising resource to be explored by the food industry.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); national funding by FCT, P.I., through the institutional scientific employment program-contract for L. Barros, R. Calhelha, M.I. Dias and C. Pereira‘s contracts; for the research contract of C. Caleja (Project HealthyPETFOOD, POCI-01-0247-FEDER-047073); and to FEDER-Interreg España-Portugal programme for financial support through the project TRANSCoLAB 0612_TRANS_CO_LAB_2_P; and to FEDER through the Regional Operational Program North 2020, under the Project GreenHealth (Norte-01-0145-FEDER-000042). This work has been supported by the Ministry of Education, Science and Technological Development of Republic of Serbia (451-03-9/2021-14/200007).info:eu-repo/semantics/publishedVersio