Phlorotannins
PRODUCT NOT FOR SALE - THIS IS A R&D RESOURCE PAGE
Phlorotannin (PT) experimental design
IDEAL PROTOCOL
Extraction and Identification of Phlorotannins from the Brown Alga, Sargassum fusiforme(Harvey) Setchell
Extracting and isolating Phlorotannin fractions from Kelp
PT- Moderately polar nature
Fig. 2. Commonly used extraction and purification methods for obtaining phlorotannins from brown seaweeds. The flowchart presents the whole extraction/ purification process from the dry alga to the isolated compound. Classical methods are shown on the left boxes and alternative methods on the right ones. In brackets are the most utilized resources (e.g., solvent, solid phase) in each technique. Circles indicate phlorotannins characterization and identification methods.
Extraction procedures:
Crude extraction can be performed in moderately polar extracts such as hydroethanolic extracts, methanol water extracts,
Purification procedures
Liquid Liquid options:
E.K. Kim et al. 2015 sued 80% methanol water extract followed by liquid liquid extract with ethyl acetate obtained PT rich extract
Liquid-liquid extraction with 80% methanol:
Ethyl acetate has been broadly employed to obtain enriched phlorotannin fractions from raw extracts (E.K. Kim et al., 2015; Li et al., 2017; S.R. Park, Kim, Jang, Yang, & Kim, 2018; R. Zhang et al., 2018). For instance, in E.K. Kim et al. (2015) ethyl acetate was used to obtain phlorotannins from an 80% methanol–water extract of Ecklonia cava, followed by the isolation of dieckol. Although ethyl acetate is a permitted flavoring and extraction solvent in the food and pharmaceutical industries, it is a toxic substance and can cause organ damage when repeatedly inhaled or ingested (TOXNET, 2015).
kim et al source + methods
https://www.mdpi.com/1660-3397/13/4/1785
SPE procedures options:
Macroporous resins, silica gel, and dispersants, are safer alternatives. Haider, Zhenxing, Hong, and Jamil (2009) demonstrated that macroporous resins are better than silica gel and polyvinylpolypyrrolidone in adsorbing/desorbing phlorotannins.
HP20 SPE - Later, J. Kim et al. (2014) reported that HP-20 is a suitable macroporous resin to purify phlorotannins and to eliminate co-extracted arsenic from E. cava (recovery: 92%, purity: 90.5%);
source plus methods:
https://www.sciencedirect.com/science/article/abs/pii/S0308814614005810
Buy HP20:
https://www.analytics-shop.com/us/su13605?utm_source=google_shopping&utm_medium=cpc&gclid=CjwKCAjwyNSoBhA9EiwA5aYlb2xHki2mNkJNUaDZVJ2SFpyev4EqFMTAI5d3dQLE0pBmyL6KMpEGHhoC5HYQAvD_BwE
XAD-16N SPE Leyton, Vergara-Salinas, Pérez-Correa, and Lienqueo (2017) found that XAD-16N is a good option to purify phlorotannins from M. pyrifera (recovery: 42%).
source plus methods:
https://www.sciencedirect.com/science/article/abs/pii/S0308814617309184
Celite and Cellulose SPE:
The capacity of phlorotannins to adsorb on cellulose and celite has also been exploited through dispersive purification steps (Ferreres et al., 2012; H.A. Lee, Lee, & Han, 2017; Sadeeshkumar et al., 2017).
Ferreres, F., Lopes, G., Gil-Izquierdo, A., Andrade, P. B., Sousa, C., Mouga, T., & Valentao, P. (2012). Phlorotannin extracts from fucales characterized by HPLC-DAD-ESI-MSn : Approaches to hyaluronidase inhibitory capacity and antioxidant properties. Marine Drugs, 10(12), 2766–2781.
MWD Molecular weight Dialysis not recommended, not scalable
2. Phlorotannins and their relevance
Phlorotannins are polyphenols unique to brown seaweeds (Phaeophyta). In contrast to terrestrial plant polyphenols, which are gallic acid and flavonoid polymers, phlorotannins are solely based on phloroglucinol (1,3,5-tri-hydroxybenzene) (Fig. 1, A). The phloroglucinol monomeric unit is synthesized via the acetate-malonate pathway and its condensation gives rise to chains- and net-like structures with diverse molecular weights: the phlorotannins (Shibata, Fujimoto, Nagayama, Yamaguchi, & Nakamura, 2002). Phlorotannin biogenesis is attributed to the Golgi apparatus and the endoplasmic reticulum, which has been seen to produce small phenolic-rich vesicles called physodes (Schoenwaelder & Clayton, 2000). Physodes are the reservoir of soluble phlorotannins, while a small fraction of insoluble phlorotannins is associated with proteins and alginates of the cell wall. The content of phlorotannins can reach up to 30% of the algae dry weight, especially in species belonging to the Fucales order. However, this is extremely variable, depending on environmental conditions (e.g., temperature, UV radiation intensity, nutrient concentration, grazing pressure) and intrinsic factors (e.g., age, thallus morphology, growth rate) (Mannino & Micheli, 2020). They are classified into six major groups (Fig. 1, B-H), according to the type of linkages between phloroglucinol units and their content of hydroxyl groups: fucols, with arylaryl linkages; phlorethols, with aryl-ether linkages; fucophlorethols, with aryl-aryl and aryl-ether units; fuhalols, with aryl-ether linkages and additional OH groups in every third ring; carmalols, with a dibenzodioxin moiety and derived from phlorethols; and eckols, with at least one three-ring moiety with a dibenzodioxin element substituted by a phenoxyl group at C-4 (K.W. Glombitza & Pauli, 2003).
Due to their polymeric structures, phlorotannins are potent free radicals scavengers; they also modulate proteins and chelate metals (Ragan, Smidsrød, & Larsen, 1979; Stern, Hagerman, Steinberg, & Mason, 1996; Wijesinghe, Ko, & Jeon, 2011). These capacities explain the wide range of cellular and ecological roles of phlorotannins in seaweeds. They are involved in cell wall hardening, accomplishing structure and reproductive functions, such as protection of the zygote, adhesion of zygotes to substrate and wound healing. Moreover, phlorotannins constitute a defense against herbivory, desiccation, high UVB radiation, and toxic heavy metals concentrations (Mannino & Micheli, 2020). For instance, it has been seen that under copper contamination,
their concentration in the cell wall increases together with their exudation to the water, preventing copper from entering and damaging the photosynthetic system (Connan & Stengel, 2011).
Available evidence states that phlorotannins not only play important ecological roles in seaweeds but also would have beneficial health effects in humans. In fact, in the last fifteen years research has mainly focused on addressing the capacity of phlorotannins to regulate relevant physiological processes that affect body functions, such as digestion, metabolism, inflammation, and cell proliferation. Antidiabetic, anticancer, antibacterial, and anti-aging are just some of the potential health benefits that have been identified (Jang et al., 2015; H.J. Kim et al., 2018; Sharifuddin, Chin, Lim, & Phang, 2015). Although several studies have involved crude brown seaweed extracts, many others were performed with purified phlorotannins or isolated compounds (Catarino et al., 2017). In this context, seaweeds from the Lessoniaceae family are the most studied, particularly the phlorotannins eckol (a phloroglucinol trimer) and dieckol (a phloroglucinol hexamer) (Fig. 1, G-H), found in high quantities in Ecklonia, Eisenia and Ishige species (Manandhar, Paudel, Seong, Jung, & Choi, 2019; Rosa et al., 2019). Even though health benefit claims are based on a large number of in vitro and animal studies, only a few clinical trials have been performed. On the other hand, investigations into the development of extraction and purification processes oriented to the obtention of high yield phlorotannins extracts or isolated phlorotannins have sharply increased, mainly those with food or pharmaceutical applications (Cikos, Jokic, Subaric, & Jerkovic, 2018).
3. Phlorotannin extraction and purification
Phlorotannins can be extracted from seaweeds by different methods. The most typical one is the traditional solid–liquid extraction (SLE) by maceration. This involves the contact of the matrix with high volumes of solvents during long periods, at room or high temperatures. In SLE methods the yield and the composition of the extracts depend on the solvent type, the solid–liquid ratio, and the extraction time and temperature (Leyton et al., 2016; Li et al., 2017). As phlorotannins are moderately polar compounds, high yields have been achieved using methanol, ethanol, acetone or their aqueous mixtures (e.g., acetone 70%), using high temperatures and long extraction times (Catarino, Silva, Mateus, & Cardoso, 2019; Wang et al., 2012). However, similar extraction yields can be achieved using alternative environmentally friendly extraction techniques. For instance, Tanniou et al. (2013) demonstrated that pressurized hot liquid extraction (PHLE) with a 75:25 ethanol–water mixture yields S. muticum extracts with high polyphenol content and antioxidant activities; they found that PHLE has a performance similar to centrifugal partition extraction (CPE) with 50:50 ethyl acetate-water and classical SLE, but with higher productivity and ecosustainability. The study also showed that supercritical CO 2 (SC-CO2 ) extraction is not suitable for the recovery of phlorotannins from brown algae. However, the addition of water and ethanol as co-solvents in SCCO 2 extraction has been shown to significantly increase the polyphenol yields obtained with pure CO 2 (Conde, Moure, & Domínguez, 2014; Saravana et al., 2017).
Other environmentally friendly processes applied to produce phlorotannin-rich extracts are microwave-assisted extraction (MAE) and ultrasound-assisted extraction (UAE). An optimized aqueous MAE procedure (solid to liquid ratio 1:30, 160 °C, 3 min) was shown to increase by 70% the phlorotannin yield and purity of the extracts, and to reduce the extraction times compared to SLE with organic solvents, due to the MAE’s ability to decompose the cellular structure according to scanning electron microscopy images (Magnusson et al., 2017; R. Zhang et al., 2018). UAE with ethanol/water mixtures enhances the phenolic content and the antioxidant capacity, as well as shortens the maceration time in brown algae extractions (Agregán et al., 2019; Dang et al., 2017). In addition, Kadam, Tiwari, Smyth, and O'Donnell (2015) reported an optimized UAE method with aqueous-HCl as solvent (0.03 M
2 F. Erpel, et al.
Food Research International 137 (2020) 109589
Fig. 1. Chemical structure of phloroglucinol and examples of phlorotannins for each of the six major groups identified to date. A: Phloroglucinol monomeric unit; B: Trifucol; C: Tetraphlorethol B; D: Fucodiphlorethol A; E: Pentafuhalol B; F: Diphlorethohydroxycarmalol; G: Eckol; H: Dieckol.
HCl, 25 min, amplitude 114 µ m), to obtain high yields of phenolic compounds, fucose, and uronic acid from Ascophyllum nodosum.
Enzyme assisted extraction (EAE) is another green approach that takes advantage of the hydrolytic activity of proteases and carbohydrases to unbound cell wall phlorotannins. It has been mainly used as a pre-treatment to enhance the phlorotannin yields of alkaline SLE procedures (Leyton, Pezoa-Conte, Mäki-Arvela, Mikkola, & Lienqueo, 2017; Siriwardhana et al., 2008).
Overall, PHLE has been the most employed, environmentally friendly technique to efficiently obtain phlorotannin-rich extracts with potential applications in functional foods or nutraceutical products (Heavisides et al., 2018; Montero et al., 2016; Sánchez-Camargo et al., 2016; Sanz-Pintos et al., 2017; Tierney, Smyth, Hayes, et al., 2013). Nevertheless, PHLE is difficult to scale-up and in some cases may be economically unfeasible for industrial production due to the high-pressure that the equipment needs to withstand to keep the solvent in the liquid state (Cuevas-Valenzuela, Vergara-Salinas, & Pérez-Correa, 2017). Finally, it should be noted that after using even the most efficient extraction procedure, a significant fraction of non-extractable phlorotannins remains in the residue of the seaweed matrix, due to their strong associations with protein or dietary fiber, and additional hydrolysis steps are required to release them (Sanz-Pintos et al., 2017).
Extraction methods recover not only phlorotannins but also pigments, alginates, and other brown algae compounds. Therefore, additional separation steps are necessary to obtain purified phlorotannins
for semi-preparative or analytical purposes. The main separation methods are liquid–liquid extraction and solid-phase-extraction (SPE) based on the polarity of the molecules, as well as dialysis based on the molecular size. Liquid-liquid extraction with ethyl acetate has been broadly employed to obtain enriched phlorotannin fractions from raw extracts (E.K. Kim et al., 2015; Li et al., 2017; S.R. Park, Kim, Jang, Yang, & Kim, 2018; R. Zhang et al., 2018). For instance, in E.K. Kim et al. (2015) ethyl acetate was used to obtain phlorotannins from an 80% methanol–water extract of Ecklonia cava, followed by the isolation of dieckol. Although ethyl acetate is a permitted flavoring and extraction solvent in the food and pharmaceutical industries, it is a toxic substance and can cause organ damage when repeatedly inhaled or ingested (TOXNET, 2015). Thus, SPE procedures, such as macroporous resins, silica gel, and dispersants, are safer alternatives. Haider, Zhenxing, Hong, and Jamil (2009) demonstrated that macroporous resins are better than silica gel and polyvinylpolypyrrolidone in adsorbing/desorbing phlorotannins. Later, J. Kim et al. (2014) reported that HP-20 is a suitable macroporous resin to purify phlorotannins and to eliminate co-extracted arsenic from E. cava (recovery: 92%, purity: 90.5%); in addition, Leyton, Vergara-Salinas, Pérez-Correa, and Lienqueo (2017) found that XAD-16N is a good option to purify phlorotannins from M. pyrifera (recovery: 42%). The capacity of phlorotannins to adsorb on cellulose and celite has also been exploited through dispersive purification steps (Ferreres et al., 2012; H.A. Lee, Lee, & Han, 2017; Sadeeshkumar et al., 2017).
3 F. Erpel, et al.
Food Research International 137 (2020) 109589
Molecular weight cut-off (MWCO) dialysis is another food-grade approach to separate phlorotannins by size and to remove interferences. For example, Tierney, Smyth, Rai, et al. (2013) and Heffernan, Brunton, FitzGerald, and Smyth (2015) fractionated low and high molecular weight phlorotannins using a 3.5 kDa MWCO membrane; the low molecular fraction (< 3.5 kDa) was then separated by reverse-phase flashchromatography to get rid of small sugars.
Further separations by preparative chromatography combined with detectors (typically ultra-violet detectors) are needed to obtain isolated phlorotannins. The standard methods are size exclusion chromatography with Sephadex LH-20 (Sadeeshkumar et al., 2016; C. Zhang et al., 2011; Zhou, Yi, Ding, He, & Yan, 2019), reverse-phase chromatography (Kirke, Smyth, Rai, Kenny, & Stengel, 2017; Yotsu-Yamashita et al., 2013), and thin-layer chromatography (Eom et al., 2012; Shibata, Yamaguchi, Nagayama, Kawaguchi, & Nakamura, 2002). They are used alone or in tandem to reach a better fractionation. For instance, by sequentially using reverse-phase C18 chromatography, Sephadex LH-20 gel filtration and thin-layer chromatography on silica gel, Eom et al. (2012) were able to isolate two phlorotannins from E. bicyclis; the compounds were later identified as fucofuroeckol A and dioxinodehydroeckol by nuclear magnetic resonance (NMR).
Fig. 2 presents a schematic representation of the general steps and the most used extraction and purification methods to obtain phlorotannin-rich extracts or isolated phlorotannins from brown seaweeds. It also shows the conventional techniques to characterize the phlorotannin content in the output product of each step, which is reviewed in the next section.
Sources
Extraction + isolation
Phlorotannins: From isolation and structural characterization, to the evaluation of their antidiabetic and anticancer potential Fernanda Erpela , Raquel Mateosb , Jara Pérez-Jiménezb , José Ricardo Pérez-Correaa,⁎ Chemical and Bioprocess Engineering Department, School of Engineering, Ponti fi cia Universidad Católica de Chile, Vicuña Mackenna 4860, P.O. Box 306, Santiago 7820436, Chile b Department of Metabolism and Nutrition, Institute of Food Science, Technology and Nutrition (ICTAN-CSIC), Calle José Antonio Novais, 10, Madrid 28040, Spain
Purification + methods
Gall, E. A., Lelchat, F., Hupel, M., Jégou, C., & Stiger-Pouvreau, V. (2015). Extraction and Purification of Phlorotannins from Brown Algae. Natural Products From Marine Algae, 131–143. doi:10.1007/978-1-4939-2684-8_7
PRODUCT NOT FOR SALE - THIS IS A R&D RESOURCE PAGE
Phlorotannin (PT) experimental design
IDEAL PROTOCOL
Extraction and Identification of Phlorotannins from the Brown Alga, Sargassum fusiforme(Harvey) Setchell
Extracting and isolating Phlorotannin fractions from Kelp
PT- Moderately polar nature
Fig. 2. Commonly used extraction and purification methods for obtaining phlorotannins from brown seaweeds. The flowchart presents the whole extraction/ purification process from the dry alga to the isolated compound. Classical methods are shown on the left boxes and alternative methods on the right ones. In brackets are the most utilized resources (e.g., solvent, solid phase) in each technique. Circles indicate phlorotannins characterization and identification methods.
Extraction procedures:
Crude extraction can be performed in moderately polar extracts such as hydroethanolic extracts, methanol water extracts,
Purification procedures
Liquid Liquid options:
E.K. Kim et al. 2015 sued 80% methanol water extract followed by liquid liquid extract with ethyl acetate obtained PT rich extract
Liquid-liquid extraction with 80% methanol:
Ethyl acetate has been broadly employed to obtain enriched phlorotannin fractions from raw extracts (E.K. Kim et al., 2015; Li et al., 2017; S.R. Park, Kim, Jang, Yang, & Kim, 2018; R. Zhang et al., 2018). For instance, in E.K. Kim et al. (2015) ethyl acetate was used to obtain phlorotannins from an 80% methanol–water extract of Ecklonia cava, followed by the isolation of dieckol. Although ethyl acetate is a permitted flavoring and extraction solvent in the food and pharmaceutical industries, it is a toxic substance and can cause organ damage when repeatedly inhaled or ingested (TOXNET, 2015).
kim et al source + methods
https://www.mdpi.com/1660-3397/13/4/1785
SPE procedures options:
Macroporous resins, silica gel, and dispersants, are safer alternatives. Haider, Zhenxing, Hong, and Jamil (2009) demonstrated that macroporous resins are better than silica gel and polyvinylpolypyrrolidone in adsorbing/desorbing phlorotannins.
HP20 SPE - Later, J. Kim et al. (2014) reported that HP-20 is a suitable macroporous resin to purify phlorotannins and to eliminate co-extracted arsenic from E. cava (recovery: 92%, purity: 90.5%);
source plus methods:
https://www.sciencedirect.com/science/article/abs/pii/S0308814614005810
Buy HP20:
https://www.analytics-shop.com/us/su13605?utm_source=google_shopping&utm_medium=cpc&gclid=CjwKCAjwyNSoBhA9EiwA5aYlb2xHki2mNkJNUaDZVJ2SFpyev4EqFMTAI5d3dQLE0pBmyL6KMpEGHhoC5HYQAvD_BwE
XAD-16N SPE Leyton, Vergara-Salinas, Pérez-Correa, and Lienqueo (2017) found that XAD-16N is a good option to purify phlorotannins from M. pyrifera (recovery: 42%).
source plus methods:
https://www.sciencedirect.com/science/article/abs/pii/S0308814617309184
Celite and Cellulose SPE:
The capacity of phlorotannins to adsorb on cellulose and celite has also been exploited through dispersive purification steps (Ferreres et al., 2012; H.A. Lee, Lee, & Han, 2017; Sadeeshkumar et al., 2017).
Ferreres, F., Lopes, G., Gil-Izquierdo, A., Andrade, P. B., Sousa, C., Mouga, T., & Valentao, P. (2012). Phlorotannin extracts from fucales characterized by HPLC-DAD-ESI-MSn : Approaches to hyaluronidase inhibitory capacity and antioxidant properties. Marine Drugs, 10(12), 2766–2781.
MWD Molecular weight Dialysis not recommended, not scalable
2. Phlorotannins and their relevance
Phlorotannins are polyphenols unique to brown seaweeds (Phaeophyta). In contrast to terrestrial plant polyphenols, which are gallic acid and flavonoid polymers, phlorotannins are solely based on phloroglucinol (1,3,5-tri-hydroxybenzene) (Fig. 1, A). The phloroglucinol monomeric unit is synthesized via the acetate-malonate pathway and its condensation gives rise to chains- and net-like structures with diverse molecular weights: the phlorotannins (Shibata, Fujimoto, Nagayama, Yamaguchi, & Nakamura, 2002). Phlorotannin biogenesis is attributed to the Golgi apparatus and the endoplasmic reticulum, which has been seen to produce small phenolic-rich vesicles called physodes (Schoenwaelder & Clayton, 2000). Physodes are the reservoir of soluble phlorotannins, while a small fraction of insoluble phlorotannins is associated with proteins and alginates of the cell wall. The content of phlorotannins can reach up to 30% of the algae dry weight, especially in species belonging to the Fucales order. However, this is extremely variable, depending on environmental conditions (e.g., temperature, UV radiation intensity, nutrient concentration, grazing pressure) and intrinsic factors (e.g., age, thallus morphology, growth rate) (Mannino & Micheli, 2020). They are classified into six major groups (Fig. 1, B-H), according to the type of linkages between phloroglucinol units and their content of hydroxyl groups: fucols, with arylaryl linkages; phlorethols, with aryl-ether linkages; fucophlorethols, with aryl-aryl and aryl-ether units; fuhalols, with aryl-ether linkages and additional OH groups in every third ring; carmalols, with a dibenzodioxin moiety and derived from phlorethols; and eckols, with at least one three-ring moiety with a dibenzodioxin element substituted by a phenoxyl group at C-4 (K.W. Glombitza & Pauli, 2003).
Due to their polymeric structures, phlorotannins are potent free radicals scavengers; they also modulate proteins and chelate metals (Ragan, Smidsrød, & Larsen, 1979; Stern, Hagerman, Steinberg, & Mason, 1996; Wijesinghe, Ko, & Jeon, 2011). These capacities explain the wide range of cellular and ecological roles of phlorotannins in seaweeds. They are involved in cell wall hardening, accomplishing structure and reproductive functions, such as protection of the zygote, adhesion of zygotes to substrate and wound healing. Moreover, phlorotannins constitute a defense against herbivory, desiccation, high UVB radiation, and toxic heavy metals concentrations (Mannino & Micheli, 2020). For instance, it has been seen that under copper contamination,
their concentration in the cell wall increases together with their exudation to the water, preventing copper from entering and damaging the photosynthetic system (Connan & Stengel, 2011).
Available evidence states that phlorotannins not only play important ecological roles in seaweeds but also would have beneficial health effects in humans. In fact, in the last fifteen years research has mainly focused on addressing the capacity of phlorotannins to regulate relevant physiological processes that affect body functions, such as digestion, metabolism, inflammation, and cell proliferation. Antidiabetic, anticancer, antibacterial, and anti-aging are just some of the potential health benefits that have been identified (Jang et al., 2015; H.J. Kim et al., 2018; Sharifuddin, Chin, Lim, & Phang, 2015). Although several studies have involved crude brown seaweed extracts, many others were performed with purified phlorotannins or isolated compounds (Catarino et al., 2017). In this context, seaweeds from the Lessoniaceae family are the most studied, particularly the phlorotannins eckol (a phloroglucinol trimer) and dieckol (a phloroglucinol hexamer) (Fig. 1, G-H), found in high quantities in Ecklonia, Eisenia and Ishige species (Manandhar, Paudel, Seong, Jung, & Choi, 2019; Rosa et al., 2019). Even though health benefit claims are based on a large number of in vitro and animal studies, only a few clinical trials have been performed. On the other hand, investigations into the development of extraction and purification processes oriented to the obtention of high yield phlorotannins extracts or isolated phlorotannins have sharply increased, mainly those with food or pharmaceutical applications (Cikos, Jokic, Subaric, & Jerkovic, 2018).
3. Phlorotannin extraction and purification
Phlorotannins can be extracted from seaweeds by different methods. The most typical one is the traditional solid–liquid extraction (SLE) by maceration. This involves the contact of the matrix with high volumes of solvents during long periods, at room or high temperatures. In SLE methods the yield and the composition of the extracts depend on the solvent type, the solid–liquid ratio, and the extraction time and temperature (Leyton et al., 2016; Li et al., 2017). As phlorotannins are moderately polar compounds, high yields have been achieved using methanol, ethanol, acetone or their aqueous mixtures (e.g., acetone 70%), using high temperatures and long extraction times (Catarino, Silva, Mateus, & Cardoso, 2019; Wang et al., 2012). However, similar extraction yields can be achieved using alternative environmentally friendly extraction techniques. For instance, Tanniou et al. (2013) demonstrated that pressurized hot liquid extraction (PHLE) with a 75:25 ethanol–water mixture yields S. muticum extracts with high polyphenol content and antioxidant activities; they found that PHLE has a performance similar to centrifugal partition extraction (CPE) with 50:50 ethyl acetate-water and classical SLE, but with higher productivity and ecosustainability. The study also showed that supercritical CO 2 (SC-CO2 ) extraction is not suitable for the recovery of phlorotannins from brown algae. However, the addition of water and ethanol as co-solvents in SCCO 2 extraction has been shown to significantly increase the polyphenol yields obtained with pure CO 2 (Conde, Moure, & Domínguez, 2014; Saravana et al., 2017).
Other environmentally friendly processes applied to produce phlorotannin-rich extracts are microwave-assisted extraction (MAE) and ultrasound-assisted extraction (UAE). An optimized aqueous MAE procedure (solid to liquid ratio 1:30, 160 °C, 3 min) was shown to increase by 70% the phlorotannin yield and purity of the extracts, and to reduce the extraction times compared to SLE with organic solvents, due to the MAE’s ability to decompose the cellular structure according to scanning electron microscopy images (Magnusson et al., 2017; R. Zhang et al., 2018). UAE with ethanol/water mixtures enhances the phenolic content and the antioxidant capacity, as well as shortens the maceration time in brown algae extractions (Agregán et al., 2019; Dang et al., 2017). In addition, Kadam, Tiwari, Smyth, and O'Donnell (2015) reported an optimized UAE method with aqueous-HCl as solvent (0.03 M
2 F. Erpel, et al.
Food Research International 137 (2020) 109589
Fig. 1. Chemical structure of phloroglucinol and examples of phlorotannins for each of the six major groups identified to date. A: Phloroglucinol monomeric unit; B: Trifucol; C: Tetraphlorethol B; D: Fucodiphlorethol A; E: Pentafuhalol B; F: Diphlorethohydroxycarmalol; G: Eckol; H: Dieckol.
HCl, 25 min, amplitude 114 µ m), to obtain high yields of phenolic compounds, fucose, and uronic acid from Ascophyllum nodosum.
Enzyme assisted extraction (EAE) is another green approach that takes advantage of the hydrolytic activity of proteases and carbohydrases to unbound cell wall phlorotannins. It has been mainly used as a pre-treatment to enhance the phlorotannin yields of alkaline SLE procedures (Leyton, Pezoa-Conte, Mäki-Arvela, Mikkola, & Lienqueo, 2017; Siriwardhana et al., 2008).
Overall, PHLE has been the most employed, environmentally friendly technique to efficiently obtain phlorotannin-rich extracts with potential applications in functional foods or nutraceutical products (Heavisides et al., 2018; Montero et al., 2016; Sánchez-Camargo et al., 2016; Sanz-Pintos et al., 2017; Tierney, Smyth, Hayes, et al., 2013). Nevertheless, PHLE is difficult to scale-up and in some cases may be economically unfeasible for industrial production due to the high-pressure that the equipment needs to withstand to keep the solvent in the liquid state (Cuevas-Valenzuela, Vergara-Salinas, & Pérez-Correa, 2017). Finally, it should be noted that after using even the most efficient extraction procedure, a significant fraction of non-extractable phlorotannins remains in the residue of the seaweed matrix, due to their strong associations with protein or dietary fiber, and additional hydrolysis steps are required to release them (Sanz-Pintos et al., 2017).
Extraction methods recover not only phlorotannins but also pigments, alginates, and other brown algae compounds. Therefore, additional separation steps are necessary to obtain purified phlorotannins
for semi-preparative or analytical purposes. The main separation methods are liquid–liquid extraction and solid-phase-extraction (SPE) based on the polarity of the molecules, as well as dialysis based on the molecular size. Liquid-liquid extraction with ethyl acetate has been broadly employed to obtain enriched phlorotannin fractions from raw extracts (E.K. Kim et al., 2015; Li et al., 2017; S.R. Park, Kim, Jang, Yang, & Kim, 2018; R. Zhang et al., 2018). For instance, in E.K. Kim et al. (2015) ethyl acetate was used to obtain phlorotannins from an 80% methanol–water extract of Ecklonia cava, followed by the isolation of dieckol. Although ethyl acetate is a permitted flavoring and extraction solvent in the food and pharmaceutical industries, it is a toxic substance and can cause organ damage when repeatedly inhaled or ingested (TOXNET, 2015). Thus, SPE procedures, such as macroporous resins, silica gel, and dispersants, are safer alternatives. Haider, Zhenxing, Hong, and Jamil (2009) demonstrated that macroporous resins are better than silica gel and polyvinylpolypyrrolidone in adsorbing/desorbing phlorotannins. Later, J. Kim et al. (2014) reported that HP-20 is a suitable macroporous resin to purify phlorotannins and to eliminate co-extracted arsenic from E. cava (recovery: 92%, purity: 90.5%); in addition, Leyton, Vergara-Salinas, Pérez-Correa, and Lienqueo (2017) found that XAD-16N is a good option to purify phlorotannins from M. pyrifera (recovery: 42%). The capacity of phlorotannins to adsorb on cellulose and celite has also been exploited through dispersive purification steps (Ferreres et al., 2012; H.A. Lee, Lee, & Han, 2017; Sadeeshkumar et al., 2017).
3 F. Erpel, et al.
Food Research International 137 (2020) 109589
Molecular weight cut-off (MWCO) dialysis is another food-grade approach to separate phlorotannins by size and to remove interferences. For example, Tierney, Smyth, Rai, et al. (2013) and Heffernan, Brunton, FitzGerald, and Smyth (2015) fractionated low and high molecular weight phlorotannins using a 3.5 kDa MWCO membrane; the low molecular fraction (< 3.5 kDa) was then separated by reverse-phase flashchromatography to get rid of small sugars.
Further separations by preparative chromatography combined with detectors (typically ultra-violet detectors) are needed to obtain isolated phlorotannins. The standard methods are size exclusion chromatography with Sephadex LH-20 (Sadeeshkumar et al., 2016; C. Zhang et al., 2011; Zhou, Yi, Ding, He, & Yan, 2019), reverse-phase chromatography (Kirke, Smyth, Rai, Kenny, & Stengel, 2017; Yotsu-Yamashita et al., 2013), and thin-layer chromatography (Eom et al., 2012; Shibata, Yamaguchi, Nagayama, Kawaguchi, & Nakamura, 2002). They are used alone or in tandem to reach a better fractionation. For instance, by sequentially using reverse-phase C18 chromatography, Sephadex LH-20 gel filtration and thin-layer chromatography on silica gel, Eom et al. (2012) were able to isolate two phlorotannins from E. bicyclis; the compounds were later identified as fucofuroeckol A and dioxinodehydroeckol by nuclear magnetic resonance (NMR).
Fig. 2 presents a schematic representation of the general steps and the most used extraction and purification methods to obtain phlorotannin-rich extracts or isolated phlorotannins from brown seaweeds. It also shows the conventional techniques to characterize the phlorotannin content in the output product of each step, which is reviewed in the next section.
Sources
Extraction + isolation
Phlorotannins: From isolation and structural characterization, to the evaluation of their antidiabetic and anticancer potential Fernanda Erpela , Raquel Mateosb , Jara Pérez-Jiménezb , José Ricardo Pérez-Correaa,⁎ Chemical and Bioprocess Engineering Department, School of Engineering, Ponti fi cia Universidad Católica de Chile, Vicuña Mackenna 4860, P.O. Box 306, Santiago 7820436, Chile b Department of Metabolism and Nutrition, Institute of Food Science, Technology and Nutrition (ICTAN-CSIC), Calle José Antonio Novais, 10, Madrid 28040, Spain
Purification + methods
Gall, E. A., Lelchat, F., Hupel, M., Jégou, C., & Stiger-Pouvreau, V. (2015). Extraction and Purification of Phlorotannins from Brown Algae. Natural Products From Marine Algae, 131–143. doi:10.1007/978-1-4939-2684-8_7
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