Extraction Of Phlorotannins Research Articles

Ecklonia radiata-derived phlorotannin extract inhibits beta-amyloid fibril formation and neurotoxicity

Ecklonia radiata-derived phlorotannin extract inhibits beta-amyloid fibril formation and neurotoxicity

Antioxidant activity of phlorotannins from brown algae

Abstract: The antioxidant activity of the phlorotannins extracted from five marine algae species (Saccharina latissima, Alaria esculenta, Laminaria digitata, Fucus vesiculosus and Ascophyllum nodosum) was studied. Three phlorotannin groups, including soluble, membrane-bound, and extracted membrane-bound phlorotannins obtained by two solvent extraction methods were investigated for their DPPH radical scavenging activity. F. vesiculosus and A. nodosum showed the highest phlorotannin yield (14.83 mg-extract/g-algae and 12.80 mg-extract/g-algae, respectively) among the five algae species. Their soluble phlorophannin (SP), membrane-bound phlorotannin (MP) and extracted membrane-bound phlorotannin (eMP) extracts all showed equal or greater DPPH radical scavenging activity than the commercial antioxidants of butylated hydroxytoluene and ascorbic acid. The antioxidant potential that combines phlorotannin yield and antioxidant activity of the MP extracts of F. vesiculosus and A. nodosum (5890 mL/g and 5278 mL/g algae, respectively) were higher than those of SP and eMP, suggesting that the MPs of F. vesiculosus and A. nodosum had great potential to be used as antioxidants. Different extraction methods also showed significantly different effects on the antioxidant activity of the phlorotannin extracts. Keywords: brown algae, phlorotannin, antioxidant activity, antioxidant, bioseparation, polyphenol, solvent extraction methods DOI: 10.25165/j.ijabe.20171006.2854 Citation: Liu X, Yuan W Q, Sharma-Shivappa R, van Zanten J. Antioxidant activity of phlorotannins from brown algae. Int J Agric & Biol Eng, 2017; 10(6): 184–191.

A comparative assessment of the activity and structure of phlorotannins from the brown seaweed Carpophyllum flexuosum

The extraction and antioxidant activity of phlorotannins from the brown seaweeds Carpophyllum flexuosum, Carpophyllum plumosum and Ecklonia radiata was investigated to identify an optimised extraction process for novel anti-oxidant extracts. Subsequently, the composition of the most active phlorotannin extracts was determined. Microwave assisted extraction (MAE) using water was the most efficient extraction process with shorter processing times and a higher purity product than obtained with any of the other methods tested. MAE resulted in the fast, effective decomposition of the cellular structure, as identified through scanning electron microscopy (SEM), and this related directly to the efficiency of extraction. Phlorotannins extracted from C. flexuosum by MAE had the strongest antioxidant activity (62.1mggallicacidequivalents(GAE)/g dw of seaweed) and more than 5.5-fold greater 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability than ascorbic acid after 7-day incubation periods at 30°C and at 60°C. Six major chemical species of phlorotannin, belonging to the fuhalol group, were identified within the MAE extract using NMR and HPLC-MS. The results confirm phlorotannins from C. flexuosum to be promising natural, bio-derived and bio-compatible antioxidants, while identifying the most effective method to extract the constituents and retain antioxidant activity.

Phlorotannin extracts from Fucales: Marine polyphenols as bioregulators engaged in inflammation-related mediators and enzymes

Phlorotannins are widely recognized as an inexhaustible family of naturally occurring molecules, with great potential in food and pharmaceutical industries. The present work explores the anti-inflammatory activity of phlorotannins from different Fucus species native from the Northern Atlantic coast, and from Fucus vesiculosus Linnaeus grown in an integrated multi-trophic aquaculture (IMTA) system, all scarcely studied regarding these compounds. Purified phlorotannin extracts were evaluated for their toxicity and anti-inflammatory potential in both cell (RAW 264.7 macrophages) and cell-free systems [lipoxygenase (LOX) inhibition and nitric oxide radical (NO) scavenging activity]. Phlorotannin content ranged between 110.28 and 288.36μgofphloroglucinol equivalents(PGE)/100mg dry purified extract (DE), wild species presenting higher content than those from IMTA. Regarding LOX, a strong correlation (r=−0.9118; p<0.0001) between the IC50 values and total phlorotannin content was found, Fucus guiryi G.I. Zardi, K.R. Nicastro, E.S. Serrão & G.A. Pearson exhibiting the best inhibitory capacity (IC50=82.10μg/mL), while F. vesiculosus from IMTA was the less effective (IC50>500μg/mL). Correlation was also observed for NO scavenging (r=−0.6363; p=0.0108). The anti-inflammatory capacity of phlorotannin extracts was further evaluated using RAW 264.7 macrophages stimulated with bacterial lipopolysaccharide, as model of inflammation. The extracts studied were not toxic at the tested concentrations (31.25–500μg/mL). F. vesiculosus from wild origin was the most effective in reducing NO in cell culture medium (IC25=56.52μg/mL), closely followed by other seaweed species under study. The IMTA species was the less effective one (IC25=317.41μg/mL). According to the results obtained, phlorotannin extracts from Fucales arise as potentially beneficial in inflammation-related conditions, effectively acting upon enzymatic and non-enzymatic inflammatory targets.

Cardioprotective Effects of a Phlorotannin Extract Against Doxorubicin-Induced Cardiotoxicity in a Rat Model.

Long-term therapy with doxorubicin (DOX) is associated with high incidence of cumulative and irreversible dilated cardiomyopathy. The goal of this study was to evaluate the cardioprotective effects and safety of a phlorotannin extract from a brown algae Ecklonia cava (Seapolynol™, SPN) against DOX-induced cardiotoxicity in a rat model. A total of 42 rats were divided into six groups: control, low-dose SPN (LDS), high-dose SPN (HDS), DOX, DOX with low-dose SPN (DOX+LDS), and DOX with high-dose SPN (DOX+HDS). Echocardiography was performed at baseline and after 6 weeks. In left ventricular (LV) ejection fraction, DOX and DOX+LDS groups showed significant decreases (P < .001), while LDS, HDS, and DOX+HDS groups showed no significant change compared with control group. In LV mass index, DOX and DOX+LDS groups showed significant increases (P < .001 and P = .013), while LDS, HDS, and DOX+HDS groups showed no significant change compared with control group. In electron microscopy of the LV wall tissue, DOX+HDS group showed markedly less impaired myofibrils and mitochondria compared with both DOX and DOX+LDS groups. On the findings in echocardiography and electron microscopy, 6-week oral administration of SPN was safe and cardioprotective in a DOX-induced rat cardiotoxicity model in a dose-dependent manner.

Extraction Optimization for Phlorotannin Recovery from the Edible Brown Seaweed Ecklonia Cava

ABSTRACTPhlorotannins, marine polyphenols from the brown seaweeds, are a promising biologically active substance. This study aimed to optimize the extraction conditions for phlorotannin recovery from the brown seaweed Ecklonia cava using response surface methodology. Ethanol concentration (X1, %), extraction temperature (X2, °C), and extraction time (X3, h) were chosen as independent variables. The dependent variables were total phlorotannin content (TPhC; Y1) and yield of phlorotannins (Y2). Estimated optimal conditions were as follows: TPhC (Y1): X1 = 95%, X2 = 80°C, X3 = 22.8 h, and yield of phlorotannins (Y2): X1 = 88.3%, X2 = 80°C, X3 = 24 h. Predicted values of dependent variables under each optimal condition were TPhC (Y1) = 570.2 mg phloroglucinol equivalents/g (mg PGE/g, dry basis) and yield of phlorotannins (Y2) = 7.8% and were similar to the experimental values. In addition, content of dieckol, which is known as an indicator of phlorotannin products, was measured. Dieckol contents of the phlorotannin extracts with the highest (565.7 mg PGE/g) and lowest (315.4 mg PGE/g) TPhC were 49.1 and 34.4 mg/g (dry basis), respectively.

Improvement in carbohydrate and phlorotannin extraction from Macrocystis pyrifera using carbohydrate active enzyme from marine Alternaria sp. as pretreatment

The commercial importance of brown seaweed has been increasing over the past decade, especially due to industries interested in the extraction of phycocolloids and, more recently, of polyphenol com ...

Extraction and Identification of Phlorotannins from the Brown Alga, Sargassum fusiforme (Harvey) Setchell.

Phlorotannins are a group of complex polymers of phloroglucinol (1,3,5-trihydroxybenzene), which are unique compounds from marine brown algae. In our present study, a procedure for extraction and enrichment of phlorotannins from S. fusiforme with highly antioxidant potentials was established. After comparison of different extraction methods, the optimal extraction conditions were established as follows. The freeze-dried seaweed powder was extracted with 30% ethanol-water solvent with a solid/liquid ratio of 1:5 at temperature of 25 °C for 30 min. After extraction, the phlorotannins were fractioned by different solvents, among which the ethyl acetate fraction exhibited both the highest total phlorotannin content (88.48 ± 0.30 mg PGE/100 mg extract) and the highest antioxidant activities. The extracts obtained from these locations were further purified and characterized using a modified UHPLC-QQQ-MS method. Compounds with 42 different molecular weights were detected and tentatively identified, among which the fuhalol-type phlorotannins were the dominant compounds, followed by phlorethols and fucophlorethols with diverse degree of polymerization. Eckol-type phlorotannins including some newly discovered carmalol derivatives were detected in Sargassum species for the first time. Our study not only described the complex phlorotannins composition in S. fusiforme, but also highlighted the challenges involved in structural elucidation of these compounds.

Extraction and Quantification of Phlorotannins from Edible Brown Algae

<abstract> This study evaluated the contents of soluble and membrane-bound phlorotannins in five brown macroalgae (, , , , and ) using two different solvent extraction methods. Extraction method A used methanol, chloroform, and ethyl acetate in sequence. Method B used methanol to obtain the crude extract first and then partitioned it in turn with n-hexane, dichloromethane, ethyl acetate, and n-butanol. and showed the highest soluble phlorotannin (SP) contents (4.66 and 4.90 mg g^-1 algae, respectively) among the five algal species by extraction method A. The highest membrane-bound phlorotannin (MP) content was observed in (68.55 mg g^-1 algae), followed by (62.58 mg g^-1 algae). The MP content of all algae was higher than the SP content, indicating the great potential for harvesting value-added products from the leftover algae powder after SP extraction. The yields of SP by extraction method B were significantly different from those achieved by extraction method A (p < 0.05). Method A was better on but method B was better on in terms of SP yield, indicating that the effectiveness of extraction methods was algal species dependent.

Identification and efficient extraction method of phlorotannins from the brown seaweed Macrocystis pyrifera using an orthogonal experimental design

The brown seaweed contains a type polyphenol compound characteristic of its species, the phlorotannins, which are produced from the polymerization of phloroglucinol units. They have been extensively studied due to their pharmacological and nutraceutical properties, but there is still a need for an optimized extraction protocol. In this study, the brown seaweed Macrocystis pyrifera was employed to determine the best conditions for extraction of phlorotannins. A set of different variables were evaluated such as the use of pre-treatment, type of solvent, drying temperature, particle size, temperature and extraction time as well as the solid/liquid ratio upon extraction. The optimal conditions for the extraction of phlorotannins were: pre-treatment with hexane, extraction with water, drying temperature 40°C, particle size below 1.4mm, at 55°C for 4h and a solid-to-liquid ratio of 1:15. Under these conditions, the concentration of phlorotannins achieved in the extract was 200.5±5.6mg gallic acid equivalent (GAE)/100g dry seaweed (DS) and total antioxidant activity of the extract of 38.4±2.9mg trolox equivalent (TE)/100g DS. Further, it was possible to identify two phlorotannins through HPLC-ESI-MS analyses: phloroeckol and a tetrameric phloroglucinol. These phlorotannins have been reported in the literature to have an antidiabetic effect and prevention of Alzheimer's disease for phloroeckol, and free radical scavenging ability and antiallergic effect for tetrameric phloroglucinol. Therefore, the extract of phlorotannins has potential as medicinal foods or therapeutics for human health applications.

Method development and validation for dieckol in the standardization of phlorotannin preparations

Phlorotannins are reported to have diverse biological properties. However, no analytical methods for the standardization of phlorotannin preparations have been reported. Herein, we developed and validated an analytical method for the determination of dieckol in phlorotannin extracts (PRT) using high-performance liquid chromatography (HPLC). The optimum HPLC conditions consisted of a Supelco Discovery C18 column stationary phase, a mobile phase (A: 15 % HPLC grade methanol in deionized water, B: methanol), UV detection at 230 nm, and a flow rate of 0.7 mL/min. The optimized chromatographic conditions were validated and exhibited good specificity and linearity (R 2 > 0.9994–1.0000). The recoveries were in the range of 100.9–102.3 %. The method had good intermediate (%RSD 1.2) and intra-day (%RSD 0.4–1.7) assay precisions. This HPLC method had good accuracy and quality in the determination of dieckol in PRT.

Valorization of the Macroalgae Sargassum Muticum by Enzymatic Hydrolysis, Interest of Surfactants to Improve the Extraction of Phlorotannins and Polysaccharides

The use of surfactants to improve enzymatic hydrolysis of the macroalgae Sargassum muticum has been investigated. Visible absorption spectroscopy has been used to quantify the solubilization of both polysaccharides and phlorotannins in the hydrolysates. After total extraction, results showed that Sargassum muticum contained 2.74% (expressed in percent of the dry weight of the algae) of phlorotannins whose 32 % were in the cell wall. This result shows that it is important to access to the parietal phlorotannins. To reach this objective, we chose the enzymatic approach for destructurating the cell wall of the algae. The use of 5% dry weight (DW - 5% by weight of hydrolyzed algae) of an enzymatic mix containing a commercial beta-glucanase, a commercial protease and an alginate lyase extracted from Pseudomonas alginovora led after 3 hours of hydrolysis to the solubilization of 2.43% DW polysaccharides and 0.52% DW phlorotannins. The use of 0.5% volume of the surfactant Triton® X-100 with 10% DW of the enzymatic mix has allowed to reaching the value of 2.63% DW of solubilized phlorotannins, that is 96% of the total phenolic content. The use of non-ionic surfactant, combined to enzymatic hydrolysis, showed an increased efficiency in disrupting cell wall and solubilizing phlorotannins in Sargassum muticum.

Application of Hansen solubility approach for the subcritical and supercritical selective extraction of phlorotannins from Cystoseira abies-marina

A new theoretical approach based on Hansen solubility parameters has been employed for the first time to select the best green solvent to extract complex bioactive molecules such as phlorotannins from brown macroalgae Cystoseira abies-marina.

고속원심분배 크로마토그래피를 이용한 감태(Ecklonia cava)로부터 Eckol의 분리 및 항염증 활성

Phlorotannins and marine algal polyphenols, including dieckol, 6,6-bieckol, phloroglucinol, phlorofucofuroeckol-A, and eckol, were isolated from brown seaweeds. These compounds have beneficial bioactivities, and Ecklonia cava has become widely used for the extraction and isolation of phlorotannins. Eckol, in particular, has been to shown to have antioxidant, anti-inflammatory, anticoagulatory, and photoprotective properties. However, due to its low abundance in weaweed, the isolation and purification of eckol are difficult. Its limited availability renders the isolation and purification of eckol labor-intensive processes. Centrifugal partition chromatography (CPC) is an efficient technique for the isolation and purification of eckol. In this study, eckol was isolated from the ethyl acetate fraction of the 70% ethanol extract of E. cava using CPC with a two-phase solvent system of a n-hexane:EtOAc:methanol:water (2:8:3:7, v/v) solution. The purity and anti-inflammatory activity of the isolated eckol were verified by high-performance liquid chromatography and by assaying lipopolysaccharide-induced inflammatory responses in an immortalized murine BV2 microglial cell line, respectively. In conclusion, CPC is a useful technique for simple and efficient isolation of eckol from E. cava.

Considerations on the use of enzyme-assisted extraction in combination with pressurized liquids to recover bioactive compounds from algae

Pressurized liquids, PLE, and enzyme-assisted extraction, EAE, have been tested to improve the extraction of phlorotannins from the seaweed Sargassum muticum. Enzymatic treatment with proteases and carbohydrases, alkaline hydrolysis and PLE with ethanol:water as extracting solvent have been studied in terms of extraction yield, total phenolic content and antioxidant activity (TEAC assay). Results demonstrated that the application of PLE alone provided the highest yields and relevant antioxidant activity. An experimental design was employed to further optimize the PLE extraction conditions; optimum parameters included the use of 160°C and 95% ethanol. Under these conditions, values of 21.9%, 94.0mg gallic acid equivalentsg−1, 5.018mg phloroglucinol equivalentsg−1 and 1.275mmol trolox equivalentsg−1 were obtained for extraction yield, total phenols, total phlorotannins and TEAC, respectively. A preliminary chemical characterization by liquid chromatography coupled to mass spectrometry provided insight in terms of the mechanisms involved in the different processes.

Comparison of different techniques for the preservation and extraction of phlorotannins in the kelp Lessonia spicata (Phaeophyceae): assays of DPPH, ORAC-PGR, and ORAC-FL as testing methods

Preservation, storage, and safe transport of samples are key factors affecting brown algal phlorotannin (phenolics) quantification, their extraction yield, and antioxidant activity. This is especially relevant when field sampling is carried out in remote areas. The objective of the present study was to evaluate and compare five preservation (frozen control, freeze-dried, silica-dried, oven-dried, and air-dried) and two extraction methods (“rapid” and “traditional” extraction) of phlorotannins in the kelp Lessonia spicata (former Lessonia nigrescens). The antioxidant power of the samples treated with the different drying methods was compared through three different in vitro antioxidant assays: 2,2-diphenyl-1-picrylhydrazyl (DPPH) and oxygen radical absorbance capacity (ORAC) methods, employing fluorescein (ORAC-FL) and pyrogallol red (ORAC-PGR) as target molecules. The results of the testing methods indicated that freeze-drying afforded the best extraction yields of phlorotannins (18–30 % higher than control using frozen material) whereas concentrations from samples dried in silica and oven averaged between 17 and 20 % lower than control. The antioxidant activity of phlorotannins measured using the DPPH test decreased significantly in samples kept dried compared to that in control, with activities varying from 65 (freeze-dried) to 21 % (air-dried). Accordingly, with ORAC-PGR and ORAC-FL indexes, sample preservation employing silica-drying or air-drying caused a high decrease of the antioxidant activity of polyphenols. No differences in both total phlorotannin concentration and antioxidant activity between the two extraction methods were found.

Extraction and Purification of Phlorotannins from Brown Algae.

The interest in the physiological roles and bioactivities of plant phenols has increased over the past decades. In seaweeds, many investigations have dealt with phenolic compounds of Phaeophyceae (phlorotannins), even though little is known so far about the ecophysiological variations of their pool or their biosynthetic pathways. We describe here a simple procedure based on the use of water-organic solvent mixtures for the extraction of phlorotannins. Crude extracts are semi-purified and fractionated by separating methods based on both the polarity and the molecular size of compounds. Phenols are then quantified by the Folin-Ciocalteu method and their radical-scavenging activity is characterized using the DPPH test. All along the purification process of phenolic compounds, the efficiency of separation is assessed by (1)H-NMR.

Screening of a Marine Algal Extract for Antifungal Activities.

Over the past few years algal extracts have become increasingly interesting to the scientific community due to their promising biological properties. Phlorotannin extracts are particularly attractive partly due to their reported antifungal activity against several yeast and dermatophyte strains.The micromethod used for the evaluation of the minimum inhibitory concentration (MIC) and the minimum lethal concentration (MLC) represents an effective and solvent-saving procedure to evaluate the antifungal activity of algae extracts. Here we describe the micromethod for determining the MIC and the MLC of algal extracts by using the example of a purified phlorotannin extract of brown algae.

Enrichment and purification of marine polyphenol phlorotannins using macroporous adsorption resins

Phlorotannins are one of the most important bioactive polyphenols; however, their purification using chromatographic methods has not been explored. Here, we studied purification of phlorotannins from the crude phlorotannin extract (CPhE) of the brown seaweed Ecklonia cava using macroporous adsorption resins. For purification of phlorotannins, four resins (HP-20, SP-850, XAD-7HP, and XAD-2) were screened. Among them, HP-20 resin showed the highest adsorption and desorption capacities. In static adsorption tests, the adsorption capacity of HP-20 increased with increasing temperature (25–45°C). Optimal conditions for the dynamic experiments can be summarized as follows: total phlorotannin content (TPhC) in loading solution: 1.5mg PGE/mL, processing volume: 4 BV, flow rate: 1mL/min, temperature: 45°C, desorption solvent: 40% ethanol solution. After purification, TPhC (452mg PGE/g) and arsenic (180μg/g) of CPhE increased and decreased to 905mg PGE/g and 48μg/g, respectively. Recovery rate of phlorotannins from CPhE was 92%.

Extraction and isolation of phlorotannins from brown seaweed Turbinaria ornata (turner) j. Agardh and its antioxidant activity

The crude extract and fractions of Turbinaria ornata collected from Mandapam coast were evaluated for their antioxidant potentials. The antioxidant potential was determined using total antioxidant capacity, total phenolic content and DPPH radical scavenging activity. The ethyl acetate fraction obtained from the crude methanolic extract by different solvent extractions exhibited prominent antioxidant activity. The ethyl acetate fraction was further fractionated with column chromatography to yield 36 fractions. All the column purified fractions were subjected to thin layer chromatography analysis and the results produce 8 fractions. The antioxidant activity of crude methanolic extract and sub fractions were evaluated. The F5 fraction exhibited higher total phenolic content and antioxidant properties. On the basis of TLC, UV-Vis and FT-IR spectral analysis F5 fraction was found to contain phenolic compounds especially phlorotannins. To the best of our knowledge, the present study is the first report on the extraction and isolation of phlorotannins and this was responsible for antioxidant activity from brown seaweed Turbinaria ornata from India.