Chemodiversity and bioactivity assessment of phlorotannins from some Phaeophyta species from the Red Sea

Phlorotannins are a unique and diverse category of secondary polyphenolic metabolites biosynthesized by brown seaweeds. They have revealed a huge potential for food and medicinal applications. In this study, five brown seaweed species were investigated: Polycladia myrica, Sirophysalis trinodis, Dictyota spiralis, Sargassum euryphyllum, and Turbinaria decurrens. All seaweed species contained considerable amounts of phlorotannins with P. myrica recording the highest content (as mg PGEs g−1 DW), followed by S. euryphyllum and then S. trinodis. The presence of phlorotannins was detected using UV–Vis spectroscopy analysis in the characterized range of 280 nm to 300 nm. All species exhibited highly significant antioxidant activity using four different techniques compared to ascorbic acid. For antidiabetic activity, the maximum α-amylase and α-glucosidase activity inhibition were recorded for P. myrica (68.79 ± 0.7%; 66.73 ± 0.5%), followed by T. decurrens (66.38 ± 0.6%; 61.34 ± 0.4%) and then D. spiralis (65.99 ± 0.5%; 60.58 ± 0.4%) compared to the acarbose standard drug (73.76 ± 0.6%; 68.38 ± 0.6%), which nominated these extracts as candidates for antidiabetic drugs. Moreover, the obtained results showed effective anti-inflammatory arthritis effects, with the maximum activity value of 86.02 ± 1.3% recorded for S. euryphyllum, followed by D. spiralis (86.23 ± 1.1%), and then P. myrica (85.94 ± 1.4%). On the other side, a low inhibition percentage of acetyl cholinesterase (AChE) activity was recorded for different phlorotannin extracts, indicating low potentiality as anti-alzheimer materials. Moreover, the P. myrica phlorotannins’ extract showed no toxicity on the WISH normal cell line by using the MTT assay. The same extract comprised a variety of phlorotannins, viz fucophlorethol, fuhalol, and phlorethol compounds, as revealed by UPLC-MS/MS analysis.


Introduction
Seaweeds have long played a role in human nutrition. They provide essential nutrients and secondary metabolites, constituting a nutritious and well-balanced diet. (Okeke et al. 2021). For the populations of the Far East, seaweed cultivation and processing is always an essential source of economic income. In contrast, western countries have recently investigated seaweeds as a source of fine biochemicals of promising industrial applications . Currently, more than 15,000 novel compounds, with validated bioactive and health promoting properties, have been isolated from seaweeds as secondary metabolites (Lopes et al. 2018). Phlorotannins compounds are mainly stored by Phaeophyta species comprising up to 12% of their total dried biomass depending on the environmental conditions of the habitat, the biological status of the species, and the method adopted for the extraction (Sathya et al. 2017). These compounds are usually accumulated in the epidermal cortex layer of brown species or may also be impregnated with the marine macroalgal cell wall (Vissers et al. 2017). Phlorotannins are characteristically identical to the tannins of terrestrial plants, although of different chemical structure. Structurally, phlorotannins are polyphenolic complexes formed by the polymerization of phloroglucinol (1,3,5-trihydroxy benzene) monomers through the acetate-malonate (polyketide) metabolic route (Targett and Arnold 1998). Phlorotannins can be assembled into four classes depending on the type of aryle-aryle (C-C) and/or aryl-ether (C- O-C) bonds formed between phloroglucinol units and the number of hydroxyl (OH) groups. These classes are: 1) phlorethols and fuhalols (by ether linkages formation), 2) fucols (by aryl-aryl linkages formation), 3) fucophlorethols (both aryl-aryl and ether linkages), and 4) eckols and carmalols (by dibenzodoxine linkage formation) (Ford et al. 2019). The type of phlorotannins linkages greatly varies between brown species and is reflected by their names. For example, Fucus species contain fucols and fucophlorethols whereas Ecklonia species contain many eckol derivatives (Allwood et al. 2020). Because of the variety of these linkages, phlorotannins can have a wide range of molecular weights, from 126 (phloroglucinol unit) to more than 100 KDa of phloroglucinol polymer units with linear and/or branched linkages (Martínez and Castañeda 2013). As the molecular weight increases by polymerization, the chemical complexity of the phlorotannins' structure increases too ). In addition, phlorotannins are highly hydrophilic compounds by virtue of several phenolic-OHs in their chemical structure, which facilitates their absorption and digestion into living organisms (Okeke et al. 2021). In brown seaweed, phlorotannins act as UV protectors and as antioxidants to prevent oxidative stress (Kang et al. 2013), defense against herbivores (as appetite suppressant) and support the structure of cell walls (Koivikko et al. 2007). Numerous marine Phaeophyta species (e.g. Hizikia fusiformis, Ecklonia cava, Eisenia bicyclis, Laminaria japonica, Ecklonia kurome, Ecklonia stolonifera, Ishige okamurae, Pelvetia siliquosa, Cystoceira compressa, Sargassum thunbergii, Fucus vesiculosus and Undaria pinnatifida) have been endorsed for their valuable phlorotannins content with multiple health-promoting biological activity (Wang et al. 2012;Kim et al. 2014;Catarino et al. 2019). In practice, the genera Ecklonia and Eisenia contain many phlorotannin derivatives with effective medical applications such as eckol, triphlorethol A, fucodiphlorethol G, dieckol, 8,8′-bieckol, phlorofucofuroeckol A, 7-phloroeckol, dioxinodehydroeckol, phloroglucinol, and phlorofucofuroeckol-B (Venkatesan et al. 2019;Catarino et al. 2022). Also, I. okamurae contains 6,6′-bieckol, phloroglucinol and DPHC (diphlorethohydroxycarmalol) (Heo et al. 2009;Yoon et al. 2009). In addition, phlorotannin oligomers of dibenzodioxin with a varied degree of polymerization (DP), sulphated phlorotannins and phenolic acids were identified in Ascophyllum nodosum (Allwood et al. 2020). Phlorotannins have been evaluated in many studies as bioactive compounds capable of modulating hazardous biochemical processes triggered by chronic diseases disorders. They have wellestablished antioxidant (Sathya et al. 2017;Hermund 2018), anti-inflammatory (Barbosa et al. 2019), anticancer (Montero et al. 2016), anti-diabetic (Lordan et al. 2013;Lopes et al. 2016) activities, and decrease hyperlipidemia-related cardiovascular issues (Okada et al. 2004). They have also been validated as having valuable anti-herbivory (Emeline et al. 2021), antimicrobial, and antiviral (anti-HIV) properties (Negara et al. 2021).
In this context, this study was conducted to investigate the content and characterization of phlorotannins from five brown seaweeds commonly abundant in the Red Sea. These seaweeds belong to different genera, i.e., Polycladia myrica, Dictyota spiralis, Sargassum euryphyllum, Sirophysalis trinodis, and Turbinaria decurrens. The antioxidant, antidiabetic, antiarthritis and anti-Alzhaimer activities of these extracts were also assessed for possible therapeutic drugs applications.

Material and methods
Chemicals All used chemicals were from Sigma Aldrich (USA) and used as specified.

Collection of the macroalgae samples
The fresh brown macroalgal samples were handpicked from the front of the National Institute of Oceanography and Fisheries (NIOF), Hurghada, Red Sea between latitudes 27° 17 ′ 13 ″ N and longitudes 33° 46 ′ 21 ″ E during winter 2020. The collected Phaeophyta samples were directly brought to the laboratory in clean plastic bags containing natural sea water. Samples were then washed carefully with tap water to remove superfluous materials and shade dried at room temperature (32 ± 3ºC). Dried seaweeds material was grounded using an electric mixer and stored at 4ºC until further analyses.

Extraction of phlorotannins
Phlorotannins were extracted from the seaweeds according to the method of Koivikko et al. (2007) with some modifications. Extraction started by adding 5 mL of hexane to each seaweed sample (1 g) to remove lipids. This step was repeated several times until all lipids were removed, and the solution was centrifuged at 1,717 × g for 3 min after each addition. The seaweed material was left in the desiccator for 2 h to remove all remaining hexane. The hexane-free seaweeds material was treated for 1 h with 50 mL of acetone (70%) containing ascorbic acid (0.3% w/v). Then the mixture was centrifuged at 1,717 × g for 6 min and the supernatant was recovered.
The previous step was repeated 4 times at the same time interval for complete extraction, and the liquid portions were collected. After that, acetone was removed in a desiccator and the residue was centrifuged at 1,717 × g for 15 min. Finally, the recovered supernatant was dried using rotary evaporator and stored at 4 °C until used.

Ultraviolet spectroscopy (UV-Vis) analysis
Phlorotannins UV-Visible spectra were recorded on a double beam spectrophotometer in a micro analytical unit, Faculty of Science, Tanta University, Egypt. The UV-visible spectra were recorded over the 190-1190 nm range using a Jenway 6800 UV-visible spectrophotometer. Then, the graph was plotted between the absorbance and wavelength. Maximum wavelength of a compound is defined as the wavelength at which the compound (phlorotannins) shows maximum absorbance (Worsfold and Zagatto 2019).

FTIR analysis
FTIR analysis (Fourier Transform Infrared Spectroscopy) was performed using Perkin Elmer Spectrophotometer system to detect the characteristic peaks and their functional groups in the range of 400-4000 cm −1 where the peak values of the FTIR were recorded (Janakiraman et al. 2011).

Determination of total phlorotannins content by DMBA assay
The content of phlorotannins in each extracted sample was estimated using 2,4-dimethoxy-benzaldehyde (DMBA) reagent (2 g per 100 mL glacial acetic acid) as described by Montero et al. (2014). Ten mg of each extract were dissolved in 1 mL methanol to form stock solution. Different volumes of this stock (0-20 μL) were taken in test tubes, and methanol was added to make a final volume of 20 μL in each test tube. Then, 10 μL of N,N-dimethylformamide (DMF) were added to each sample. An aliquot of 1.25 mL HCL (16%) was added and mixed for 1 min, then, 1.25 mL of DMBA reagent was added to each tested sample. The tubes were covered and placed in 30 °C water bath for 60 min. After that, the absorbance of the developed red color was measured at 510 nm using a SpectraMax M2 e plate-reader device (USA). A calibration curve was prepared following the same procedures using phloroglucinol as standard at serial dilutions of 5-100 µg mL −1 . The results were expressed as milligram phloroglucinol equivalent (PGEs) per gram sample dry weight (DW).

Determination of total phlorotannins content by Folin-Ciocalteu assay
Total phlorotannins content was quantified according to the Folin-Ciocalteu method (Wang et al. 2012). 300 μL of each extract were mixed with 1 mL Folin reagent (10%). After keeping the mixture for 5 min, 2 mL of sodium carbonate solution (10%) were added. Then, the samples were incubated for 1.5 h in the dark at room temperature. Thereafter, the absorbance of the samples was measured at 750 nm using UV/vis spectrophotometer and phloroglucinol as standard. The results were expressed as milligram of PGEs per g dry weight of sample.

Assessment of the biological activities of phlorotannins extracts from the seaweed species
Antioxidant activity assays 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay The DPPH radical scavenging activity was determined according to Wang et al. (2008). The sample solutions were prepared by adding 1,000 μL of the tested phlorotannins to 3 mL of DPPH solution. The DPPH solution was replaced by 3 mL of methanol for preparing blank sample solution. Control sample was prepared similar to the blank but the extract was replaced by DPPH. The mixtures were vortexed for 1 min and then left to stand for 30 min in the dark at 30 °C. After that, the absorbance was measured at 550 nm. DPPH free radical scavenging activity was calculated as percentage using the following equation: where: A sample = absorbance of the sample solutions; A control = absorbance of the control samples, and A blank = absorbance of the blank.

Total antioxidant capacity assay
Total antioxidant capacity (TAC) of the extracts was determined following Prieto et al. (1999). 100 μL of the extracted phlorotannins were mixed with 3 mL of reagent (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate), and then 900 μL of distilled water were added. The samples were incubated for 90 min in a water bath at 95 °C. The absorbance of samples was measured at 695 nm, and ascorbic acid was used as standard. The results of TAC 1 3 were expressed as µg ascorbic acid equivalents (AAEs) per gram dry weight of sample.

Reducing power assay
Reducing power (RP) of the phlorotannins extracts was measured following Zhu et al. (2002). 500 μL of each extract were mixed with 0.5 mL of 0.2 M phosphate buffer (pH 7.2) and 0.2 mL of 1% potassium ferricyanide (K 3 [Fe(CN) 6 ]). After 20 min incubation at 50 °C, the following solutions were added and mixed: 500 μL of 10% trichloroacetic acid (CCL 3 COOH), 300 μL distilled water and 80 μL of 0.1% FeCL 3 . Absorbance of the developed color was measured at 655 nm. Ferrous sulfate solution was used as standard, and the results were expressed as microgram of FeSO 4 equivalents (FSEs) per g dry weight of sample.

Hydrogen peroxide radical scavenging assay
The ability of the phlorotannins extracts to scavenge H 2 O 2 radicals was assayed according to Gulçin et al. (2004). 10 mM of H 2 O 2 solution were prepared in 0.1 M phosphate buffer saline at pH 7.4. Then, 1 mL of the extract was rapidly mixed with 2 mL of the H 2 O 2 solution. After 10 min incubation at 37ºC, the absorbance was measured at 230 nm using UV-spectrophotometer against a blank without hydrogen peroxide. The scavenging of H 2 O 2 radical was calculated as percentage using the following formula: where: A 0 = absorbance of control and A 1 = absorbance of sample.

Inhibition of α-amylase activity
The anti-diabetic activity by inhibition of α-amylase, in vitro, was examined according to Hamdan and Afifi (2004) with some modifications. Test sample (500 μL) and serial dilutions of standard drug acarbose (100-1000 μg mL −1 ) were added to 500 μL phosphate buffer (0.2 mM, pH 6.9) containing α-amylase (0.5 mg mL −1 ) enzyme. The mixture was incubated at 25 °C for 10 min. before 500 μL of starch solution (1% w/v in 0.02 M sodium phosphate buffer, pH 6.9) were added to the contents, then the mixture was re-incubated at 25 °C for 10 min. The reaction was then quenched by adding 3,5-dinitrosalicylic acid reagent (1.0 mL) and heated in a boiling water bath for 5 min, then cooled at room temperature. The reaction mixture was then diluted to 10 mL with dist. H 2 O. Finally, the absorbance was measured at 540 nm for samples and calibration curve solutions. A plot of α-amylase activity inhibition was made and the IC 50 value (µg mL −1 ) was calculated from the non-linear regression curve. The IC 50 value is the concentration of the extracts/standards that inhibited 50% of the enzyme activity.

Inhibition of α-glucosidase activity
The α-glucosidase inhibition assay was performed according to Dong et al. (2012) with modification. In brief, a solution of starch substrate (2% w/v sucrose, 1 mL in 0.2 M Tris buffer, pH 8.0) and various concentrations of phlorotannins samples were incubated for 5 min at 37 °C. The reaction was initiated by adding α-glucosidase enzyme (1 mL, 1 Unit mL −1 ) to the reaction mixture, followed by incubation for 10 min at 37 °C. The reaction was stopped by adding 3,5-dinitrosalicylic acid reagent (1 mL) during heating for 5 min, cooled at room temperature, and then diluted to 10 mL with dist. H 2 O. The absorbance was measured at 540 nm as previously described and the IC 50 value (µg mL −1 ) of α-glucosidase activity was calculated.

In vitro Anti-arthritis activity
The anti-arthritis activity was evaluated using albumin denaturation test according to Singh and Sharma (2016) with some modifications. Two fold serial dilutions (ranging from 1000-10 μg mL −1 ) of different phlorotannin extracts were placed in a 96-well plate. 100 µL of the samples and 100 µL of 1% bovine albumin solution were then added to each well. Then, the plates were incubated at 28˚C for 15 min. After that, the plates were kept at 70˚C for 10 min to induce denaturation, and then cooled at room temperature. The developed turbidity in the plates was measured using microtiter plate reader (BioTek Instruments, Inc., USA) at 660 nm. Diclofenac sodium was used as standard drug in a concentration range of 1000-10 μg mL −1 and treated similarly as the test extracts to prepare a calibration curve. The percentage of protein denaturation inhibition was calculated using the following equation.
where: A 1 = absorbance of control sample (no drug was added); A 2 = absorbance of test sample or standard sample with albumin solution.
In addition, the IC 50 value was estimated as the concentration that inhibited 50% of protein denaturation under the assay conditions.

Acetyl Cholinesterase (AChE) inhibitory assay
Acetyl cholinesterase inhibitory activity of the samples was determined by Ellman's microtiter plate assay. Briefly, 140 µL of 0.1 M sodium phosphate buffer (pH 8) were first added to each well of 96-well microplate, followed by 20 µL of the samples and 20 µL of 0.09 unit mL −1 of AChE enzyme. After pre-incubation at room temperature for one min, 10 µL of 10 mM DTNB (5,5'-dithio-bis-2-nitrobenzoic acid) reagent were added into each well, followed by 10 µL of 14 mM acetylthiocholine iodide solution as a substrate. The same procedures were used for Donepezil compound as the reference standard. After 30 min of the enzymatic reaction initiation, the absorbance of the colored end-product was measured using a microtiter plate reader instrument at 412 nm. A set of eight serial concentrations was used to estimate the 50% inhibitory concentration (IC 50 ). Percentage of inhibition was calculated using the following formula (Obregon et al. 2005):

Ultra Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS) analysis
UPLC-MS/MS is used for many applications that require very high sensitivity and selectivity such as drugs and pharmaceutical formulations. Chromatographic analyses of the most active phlorotannins samples were performed on a XEVO TQD triple quadruple instrument (Waters Corporation, USA). This assay was carried out according to the following acquisition conditions: Column: ACQUITY UPLC-BEH C18 1.7 μm-2.1 × 50 mm column; flow rate: 0.2 mL min −1 ; solvent system: water containing 0.1% formic acid and methanol containing 0.1% formic acid; capillary voltage of 3 kV; temperature of 440 °C, and the mass range was set from 100 to 1000.

In vitro cytotoxicity test using cell viability MTT assay
The WISH (Wistar Institute Susan Hayflick) is a HeLa derivative human cervix carcinoma cell line, which was originally derived from human amnion tissue. The cells were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 1% L-glutamine, HEPES buffer and Inhibation % = absorbance of control − absorbance of extract absorbance of control × 100 50 µg mL −1 gentamycin. All cells were maintained at 37ºC in a humidified atmosphere with 5% CO 2 and were sub-cultured two times a week. For the cytotoxicity assay; the cells were seeded in a 96-well plate (Falcon, USA) at 1 × 10 4 cells per well in 100 µL of growth medium. Fresh medium containing different concentrations of the test samples was added after 24 h of seeding. The microtiter plates were incubated at 37ºC in a humidified incubator with 5% CO 2 for a period of 24 h. Three wells were used for each concentration of the test sample. Control cells were incubated without test sample and with or without DMSO. After incubation, viable cells were evaluated by a colorimetric method using MTT stock solution (5 mg of MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide] in 1 mL of PBS) (Mosmann 1983). The optical density (OD) was measured at 590 nm with a microtiter plate reader instrument (SunRise, Tecan Inc, USA) and the percentage of cell viability was calculated as: where: OD t is the mean optical density of wells treated with the tested sample, and OD c is the mean optical density of untreated cells.
The relation between surviving cells and sample concentration is plotted to get the survival curve of the cell line after treatment with the tested compound. In addition, the inhibitory concentration (IC 50 ), that causes toxic effects to 50% of intact cells, was estimated from the graphical plots of the dose response curve (Mosmann 1983) using Graphpad Prism 7 software (USA).

Statistical analysis
All experiments were done in tri-analytical replicates (n = 3). Data analysis used Microsoft Excel 2010 and GraphPad Prism 7 software, and values were expressed as mean ± standard deviation (SD). Statistical analysis was done using analysis of variance (ANOVA) test, SPSS package for Windows10 (IBM SPSS Statistics for Windows, Version 27.0., IBM Corp. USA) P values equal to or less than 0.05 level of significance were considered significant.

Identification of the macroalgae samples
The collected seaweed samples were identified according to Aleem (1978), Oza and Zaidi (2001)

Characterization of the extracted phlorotannins
The UV spectra of the extracted phlorotannins are shown in Fig. 1. The peaks were clear at a maximum wavelength  . 2) indicated the presence of many functional groups in the phlorotannin extracts depending on the seaweed species. In general, varied vibration intensities were noticed for the five studied seaweeds whereby P. myrica and D. spiralis exhibited the highest band intensities while S. trinodis, S. euryphyllum, T. decurrens were of moderate intensities. The results showed broad stretching bands around 3437-3371 cm −1 in all extracted phlorotannins, which indicated the presence of phenol hydroxyl groups (

Antioxidant activities
All the phlorotannin extracts showed highly significant scavenging activity of DPPH free radical expressed as percentage compared to ascorbic acid (Fig. 3). This activity was dependent on the concentration of the assayed phlorotannins. At 1000 µg mL − Concerning the total antioxidant activity (TAC) of the studied phlorotannins, the results shown in Fig. 4 presented gradual increase in the TAC with increasing the concentration of the extracts. At 1000 µg mL −1 concentration, P. myrica phlorotannins showed a non-significant TAC value of 30.28 µg AAEs g −1 dry weight compared to ascorbic acid value of 31.29 µg AAEs Following the same pattern, the reducing power capacity of seaweed phlorotannins was highly significant (Fig. 5). At 1000 µg mL −1 concentration, P. myrica, followed by S. trinodis phlorotannins demonstrated highly significant reducing power capacity of 13.81 and 11.91 µg FeSO 4 equivalents (FSEs) g −1 dry weight compared to ascorbic acid of 5.98 FSEs g −1 DW. S. euryphyllum and D. spiralis showed lower but significant reducing values of 7.97 FSEs g −1 DW and 7.91 FSEs g −1 DW, respectively, while T. decurrens displayed a non-significant reducing value of 6.18 FSEs g −1 DW.
In addition, all the tested phlorotannin extracts and standard exhibited weak scavenging capacity for hydrogen peroxide radical (Fig. 6). However, according to the results in Fig. 6, ascorbic acid scavenging activity of H 2 O 2 radical was significantly higher than that of the phlorotannin extracts at each tested concentration. At 1000 µg mL −1 concentration, P. myrica phlorotannins showed 26.44% of H 2 O 2 antioxidant activity, followed by D. spiralis (25.46%), T. decurrens (25.44%), S. trinodis (24.96%), and finally S. euryphyllum (20.96%). The IC 50 values were not evaluated for this assay since they were not detected in the tested concentrations range (100-1000 µg mL −1 ).

In vitro anti-acetyl cholinesterase (AChE) activity
The phlorotannins extracted from different brown species exhibited weak acetyl cholinesterase (AChE) inhibition activity (Tab le Fig. 5 Total reducing power as mg FeSO 4 equivalents (FSEs) per g dry weight of brown seaweed phlorotannins at different concentrations against ascorbic acid. Values are means ± SD of three replicates and asterisks * and ** refer to significant and highly significant difference at p = 0.05 level of significance Fig. 6 Hydrogen peroxide antioxidant activity as percentage of brown seaweed phlorotannins at different concentrations against ascorbic acid. Values are means ± SD of three replicates and asterisks * and ** refer to significant and highly significant difference at p = 0.05 level of significance 1 3 2). Low percentage inhibition of the enzyme activity indicated low potential capability of the extracted phlorotannins as anti-Alzheimer agents. Although having 100% anti-AChE activity at higher concentration (500 µg mL −1 ), this percentage activity could be achieved by the Donepezil standard drug at much lower concentration of 62.5 µg mL −1 . This was also reflected on the evaluated IC 50 values, which were of highly significantly different at p ≤ 0.05 level from those recorded for the standard drug.

Cytotoxicity test of phlorotannins extracted from P. myrica
Phlorotannin material isolated from P. myrica was further examined for its cytotoxicity on WISH normal cell in light of its estimated activities in the presented work. The extract was entirely safe up to 31.25 g mL −1 (Table 4). The IC 50 value could not be determined at these experimental dosages since only 20.57% of the cells were inhibited at the maximal experimental concentration (500 g mL −1 ).

UPLC-MS/MS analysis of phlorotannins extracted from P. myrica
As shown in Fig. 7, the UPLC/MS analysis of P. myrica phlorotannins reveled 12 peaks in the ES positive mode (Table 5) and 15 peaks in the ES negative one ( Table 6). Most of these peaks were tentatively assigned to phlorotannin compounds based on their measured MS spectrum and their MS/MS (m/z) fragmentation pattern (Martínez and Castañeda 2013;Nair et al. 2019). The characteristic neutral loss of tandem MS established that these molecules belong to different class of phlorotannins, and some of their tentative structures were exemplifies in Fig. 8.

Discussion
Phlorotannins are the major phenolic compounds found in brown seaweeds with a variety of chemical structures and bioactivities. The investigated brown algal species in the present study showed significant variation in phlorotannins yield and content. In this connection, Connan et al. (2004) noted that pholortannins content exhibited spatial and temporal variation between different brown algal species. The difference in phlorotannins content between the two assays, according to Stern et al. (1996), was explained by the fact that the DMBA reagent only reacts with m-diphenolics, but the FC-reagent also reacts with both mono-and O-diphenolics. Therefore, the difference in phlorotannin content represented the amount of non-phloroglucinol phenolics or fuhalol-type tannins. Fuhalol units within the phloroglucinol oligomers have an extra OH group (i.e., do not bear a m-diphenolic group) and so they do not react with the DMBA reagent (Vissers et al. 2017;Lopes et al. 2018). In addition, monomeric phloroglucinol is mostly used as a standard in both assays owing to the deficiency of phlorotannins polymeric standards. Therefore, as phloroglucinol reacts stronger to the DMBA assay reagent than phlorotannin oligomers this resulted in underestimation of the content (Stern et al. 1996).
Results of UV-vis spectrum showed λ max at 295 ± 1 nm of the phlorotannin extracts of different seaweeds indicating the presence of phenolic compounds (Fig. 1). Similar λ max values were reported by Koivikko et al. (2007) from F. vesiculosus. Likewise, Gheda et al. (2021) recorded λ max at 295 nm for phlorotannins extracted from Cystoseira compressa. However, the lack of validated standards hindered identification of oligopolymeric phlorotannins. For phloroglucinol monomer standard, the maximum absorbance was observed at 270 nm (Koivikko et al. 2007).
FT-IR analysis demonstrated several functional groups for the phlorotannin extracts of the tested brown species (Fig. 2). Broad stretching absorption bands at 3437-3371 cm −1 confirmed the presence of phenolic hydroxyl groups (OH) (Coates 2006). Absorption bands at 2952-2930 cm −1 were attributed to alkyl groups (C-H) where aromatic and nonaromatic C-H bond alteration occurred near 3000 cm −1 (Soto et al. 2005). According to Zhang et al. (2017), the presence of carbonyl groups (C = O) of carboxylic acids was demonstrated by the sharp peaks around 1772 -1718 cm −1 . The presence of C-C stretching peaks around 1619-1639 cm −1 may be attributed to C 4 -C 8 interflavonoid linkages (Oo et al. 2009). Furthermore, the broad bands at 1400 cm −1 detected in all phlorotannin extracts may be corresponding to the aromatic ring stretching vibration (Zhang et al. 2017). The presence of C-O-C, including aromatic C-O and aliphatic C-O, was indicated by peaks around 1038-1078 cm −1 in all extracts (except in D. spiralis). In addition, detection of bands at 890-660 cm −1 denoted vibration of benzene rings C-H and alcohol O-H in the extracted phlorotannins (Coates 2006).
The antioxidant activity of the extracted phlorotannins from the studied seaweed species was estimated by four different assays. The results indicated that DPPH scavenging activity differed not only according to the investigated seaweed species but also according to the structure and concentration of the extracted phlorotannins. The effect of an antioxidant on DPPH radical scavenging is owing to its hydrogen-donor ability when used as a DPPH substrate. As a stable free radical of violet color, DPPH accepts an electron or hydrogen radical and converts to a stable diamagnetic (non-radical) molecule of pale yellow (Molyneux 2004). This may explain the obtained results of the antioxidant activity and IC 50 of the studied phlorotannin extracts, which were comparable to ascorbic acid standard results. In this connection, the phlorotanninenriched ethyl acetate fraction from F. vesiculosus had the highest DPPH scavenging activity and reducing power when compared to the crude extract and different polarity fractions (Wang et al. 2012). Moreover, phlorotannins content and their antioxidant activity in seaweeds have been reported to vary depending on the species, the geographic region and the extraction method used (Jégou et al. 2015). Notable DPPH scavenging activity was reported by Liu et al. (2017) using phlorotannins extracted from five marine brown species viz. Alaria esculenta, Ascophyllum nodosum, Fucus vesiculosus, Laminaria digitata, and Saccharina latissima. As reported by Sathya et al. (2017), crude methanolic extract, F1 fraction, from C. trinodis showed 70.44% DPPH radical-scavenging activity, followed by the dichloromethane (DCM) fraction of 69.62% and compared to the values of standards antioxidant BHT of 69.27% and ascorbic acid of 57.28% Depending on solvent and fraction, the highest TAC values was observed by Sathya et al. (2017) in S. trinodis F2 fraction (180.89 µg AAE g −1 extract DW), followed by the DCM fraction (155.10 µg AAE g −1 ), and then the F5 fraction (124.23 µg AAE g −1 ). Likewise, the enzyme-assisted phlorotannins extract from Sargassum duplicatum gave a total antioxidant activity of 11.17 µg AAE g −1 DW (Boi et al. 2020). Many studies estimate the antioxidant capacity of phlorotannins using different assays. The findings showed that the branching type and accessibility of the hydroxyl groups in the phlorotannin molecules have a greater impact on their antioxidant activity than the degree of polymerization 1 3 (Hermund 2018). Numerous types of purified phlorotannins from E. cava exhibited notable antioxidant activity and were able to protect cells against H 2 O 2 -induced damage (Kang et al. 2013). As stated by Sathya et al. (2017), polyphenol are electron-rich molecules and thus can enter into efficient electron-donation reactions in the presence of oxidizing agents to form phenoxyl radical intermediate species. Phenoxyl radicals can be stabilized by delocalization of the unpaired electron around the ortho and para positions of the phenol ring. Additionally, phenoxyl radicals can also become stable by hydrogen bond interaction with an adjacent hydroxyl group. Therefore, the antioxidant activity of phlorotannins can be the result of particular scavenging of radicals formed through peroxidation, scavenging of oxygen containing compounds, or metal-chelating ability (Venkatesan et al. 2019). However, Emeline et al. (2021) stated that oxidative stress, herbivory practices, and UV-radiation are the key verified stimulants for the production of phlorotannins as secondary metabolites. In the food industry and medical applications, natural derived antioxidants like phlorotannins are considered safe for human health and valuable to producers (Okeke et al. 2021;Catarino et al. 2022). The results in Table 2 indicated a potent antidiabetic activity of the studied phlorotannins. This activity was dependent on the seaweed species, phlorotannin concentration and their chemical composition. The obtained results were in agreement with many previous studies that suggested seaweed phlorotannin extracts as a valuable source for antidiabetic drugs. As already known, α-glucosidase and α-amylase enzymes are essential for the digestion of dietary complex carbohydrates. Inhibition of these two enzymes in type 2 diabetes mellitus (DMT2) patients can slow down Fig. 8 Chemical structure of the commonly identified phlorotannins in P. myrica extract. The structure was depending on the MS/MS fragments (m/z) as adapted from Martínez andCastañeda 2013, Li et al. 2017;Vissers et al. 2017;Lopes et al. 2018;Catarino et al. 2019;and Chouh et al. 2022 the digestion of oligosaccharides and disaccharides, delay glucose absorption into blood and reduce glucose levels in plasma (Lopes et al. 2016). This will eventually lead to the conquest of postprandial hyperglycemia. Okada et al. (2004) showed that eckol, dieckol, and 7-phloroeckol phlorotannin derivatives isolated from E. bicyclis had a powerful α-amylase inhibitory capacity of ˃ 87%, at a concentration of 1 mM. The inhibitory capacity of these compounds was positively dependent on their molecular weight, and the number of free OH groups. Lee et al. (2009) reported that dieckol phlorotannin derivative of E. cava could significantly inhibit intestinal α-glucosidase (IC 50 = 10.79 µM) and pancreatic α-amylase (IC 50 = 124.98 µM). The inhibitory activity of dieckol was dose dependent and the inhibition pattern was through a non-competitive mechanism. Moon et al. (2011) described six active phlorotannin types from E. stolonifera and E. bicyclis, which exhibited strong activity against α-glucosidase compared to acarbose (Glucobay) as a synthetic glucosidase inhibitor reference drug. Other examples of reported non-toxic phlorotannins were DPHC (diphlorethohydroxycarmalol) isolated from I. okamurae (Heo et al. 2009), and DDBT [2-(4-[3,5-dihydroxyphenoxy]-3,5-dihydroxyphenoxy) benzene-13,5-triol] isolated from Sargassum patens (Kawamura-Konishi et al. 2012), which showed good α-glucosidase and α-amylase inhibitory capacity. However, the solvent used for phlorotannin extraction influences their bioactivity. Methanol phlorotannin extracts from A. nodosum showed more activity (IC 50 = 44.7 µg mL −1 ) in contrast to α-amylase, while the extract of F. spiralis proved better influence (IC 50 = 0.32 µg mL −1 ) against α-glucosidase (Lordan et al. 2013). Recently, polyphenol-rich acetone extract of Turbinaria decurrens (Abdel-Karim et al. 2021) and phlorotannins extract from C. compressa (Gheda et al. 2021) exhibited antidiabetic activities in induced diabetic rats by decreasing, α-amylase, α-glucosidase, and serum glucose activities due to increasing serum insulin and antioxidant levels. Currently, the ability of phlorotannins to combat diabetes-related disorders has been verified so that a phlorotannins-composite commercial product is already being marketed (InnoVactive. InSea2. 2021). This product is a hot water extract containing phlorotannins derived from A. nodosum and F. vesiculosus with efficient activity to inhibit both α-amylase and α-glucosidase digestive enzymes without gastrointestinal upset. The product also could induce 48% reduction in hyperglycemia levels, 12% enrichment in insulin secretion, and 8% enhancement in insulin sensitivity with no cytotoxicity syndromes. In this context, P. myrica phlorotannins derived in this study seems promising towards antidiabetic pharmacological interventions.
Furthermore, the obtained results revealed that phlorotannin extracts of the studied brown seaweeds attained a significant anti-arthritis capacity compared to the Diclofenac sodium standard drug (Table 3). The term arthritis includes about 150 rheumatic disorders related to joint. It is an eminent inflammatory joint illness, being accompanied by main structural and biochemical variations in the joint tissues ). Generally, arthritis illnesses can be divided into low-inflammatory syndromes, for example osteoarthritis and posttraumatic arthritis; and high-inflammatory syndromes, like rheumatoid, gout and psoriatic arthritis (Hazes and Luime 2011). Numerous categories of anti-inflammatory drugs are commonly available to treat the inflammatory-linked diseases, though associated with side effects. Recently, much effort has been paid to seek natural marine-based compounds as anti-inflammatory drugs (Barbosa et al. 2019). Limited studies have authorized phlorotannins from brown seaweed to treat inflammatory diseases. In a leading study for the managing of inflammatory chronic joint disease, Shin et al. (2006) validated the ability of phlorotannin-rich extracts from E. cava to reduce prostaglandin2 (PGE2) production in liposaccharide (LPS)-treated RAW 264.7 cells. The authors reported significant antioxidant activity of these extracts and their significant capacity to inhibit degradation of human recombinant IL-1α-stimulated proteoglycan in rabbit cartilage cell culture. The study thus supported the potential therapeutic use of these phlorotannin-rich extracts for modulating the pathophysiological routes of osteoarthritis. A variety of phlorotannins in E. cava extracts have been shown to inhibit mRNA gene expression and activity of the matrix metalloproteinases (MMP) as a mediators of cartilage degradation. Furthermore, they could attenuate nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2)-linked inflammatory reactions in human osteosarcoma (MG-63) by down-regulating of inflammatory response factor mitogenactivated protein kinases (MAPK) signaling pathway ). The authors suggested these compounds as valuable candidates for chronic articular diseases treatment. Wijesinghe et al. (2012) found that a fermented phlorotannins-rich extract from E. cava showed effective anti-inflammatory activities by inhibiting nitric oxide (NO) and PGE 2 production in LPSstimulated RAW 264.7 cell lines. Also, the same extract was able to suppress inducible iNOS, COX-2 expressions and the release of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in a dose-dependently manner. Similar results were obtained by Kim et al. (2014) using the same E. cava extract, in vitro and in vivo, on LPS-induced inflammation in a zebra fish model. Accompanied by the reduction in proinflammatory cytokines, Yang et al. (2016) demonstrated a reduction in the pro-inflammatory cytokine mediators of TNF-α and HMGB1 in a sepsis treatment model using E. cava phlorotannin extract. The extract also caused inhibition of the NF-κB pathway and the Nrf2/HO-1 pathway alteration as well. Likewise, phlorofucofuroeckol A and trifuhalol phlorotannins also showed a similar mechanism of decreasing NO and PGE 2 formation by reducing iNOS and COX-2 levels and down regulation of pro-inflammatory IL-1β, IL-6, and TNF-α production, and also delimited the iNOS and COX-2 expressions and controlling of NF-κB and MAPK actions (Kim et al. 2014;Phasanasophon and Kim 2019). Many of investigated phlorotannins from different seaweeds have provided anti-inflammatory activities in a more or less similar way, as for example, extracts of the genera Ecklonia, Eisenia, Ishige, Fucus, Ascophyllum, and Sargassum (Mahmoud et al. 2021). As mentioned by Chouh et al. (2022), the ethyl acetate phlorotannins fraction of S. vulgare demonstrated potent anti-inflammatory activity by superior preventing of albumin denaturation than the standard non-steroidal ketoprofen medication. In this connection, phlorotannin extracts from P. myrica, S. euryphyllum, D. spiralis and S. trinodis, in the presented study indicates that they valuable candidates as antiinflammatory drugs.
In addition, the present study recognized weak AChE ability of the phlorotannins extracted from the various studied seaweeds. Persistent and long-lasting inflammation is a source of many chronic disease including cancer, cardiovascular disease, diabetes, rheumatoid arthritis conditions, and so on (Barbosa et al. 2019). Inflammation that occurs round the central nervous system (CNS) induces neurodegeneration, mental deterioration, and Alzheimer's disease. As a neurodegenerative disease, Alzheimer's disease is defined by gradual memory fading and thinking skills weakening, with cortex-specific pathology signs in individuals elder than 65 years old (Kang et al. 2013). There are many mechanisms that describe the pathways of Alzheimer's disease. It may be caused by the reduction in the neurotransmitter acetyl choline (cholinergic hypothesis), production of β-amyloid (Aβ amyloid hypothesis), activation of tau protein abnormalities (Tau hypothesis), or by low functioning of blood brain barrier (Jude and Gopi 2021). In a former study, the inhibitory effects of three phlorotannins isolated from I. okamurae on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities was assessed by Yoon et al. (2009). 6,6′-bieckol showed non-competative inhibition kinetics type against AChE with IC 50 value of 46.42 ± 1.19 μM. Furthermore, diphlorethohydroxycarmalol exhibited a moderate inhibitory activity towards BChE with an IC 50 of 110.83 ± 1.15 μM. The authors recommended these compounds as functional food ingredients or nutraceuticals for Alzheimer disease prevention. Kang et al. (2013) conclude that E. cava ethanol extract regulates γ-secretase and α-secretase expression and activity. This caused a leading reduction in β-amyloid (Aβ), a major pathogenic peptide in Alzheimer's disease, production by the stable cells. Neuronal cell progressive damage and loss in Alzheimer's disease can be also triggered by oxidative stress and its associated reactive oxygen species (ROS). According to Lee et al. (2018), eckol, dieckol, and 8,8′-bieckol were efficient as neuroprotective candidates for Alzheimer's disease drugs. These phlorotannins, especially dieckol, were able to ameliorate intracellular oxidative stress alongside with anti-apoptotic and anti-inflammatory activities against Aβ 25-35 -induced impairment in PC-12 (pheochromocytoma) cells. Similarly, phlorotannin-rich extract from E. cava (PEEC) demonstrated neuroprotective effects counter to oxidative damage in PC-12 as well as in human (neuroblastoma) SH-SY5Y cells (Nho et al. 2020). The PEEC compound could also repress both AChE and BChE activities in a dose-dependent manner. Recently, a phlorotannin dibenzodioxin-fucodiphloroethol (DFD) from E. radiata was isolated and identified by Shrestha et al. (2021). The compound showed a protective action contrary to the aggregation of neurotoxic β-amyloid protein Aβ 1-42 and AChE activity, besides, an intracellular ROS scavenging activity in PC-12 cells. In the light of these previous studies, the phlorotannin extracts recovered from the presented study, especially from P. myrica, S. euryphyllum and T. decurrens, are worthy of further investigation as anti-Alzheimer disease agents. All of the extracts were able to achieve 100% of AChE inhibition at higher concentrations.
The cytotoxicity analysis was performed to ensure the safe use of P. myrica phlorotannins extract for future in vivo studies. As stated by Okeke et al. (2021), a certain substance is considered safe or nontoxic if its contradictory effects on physiological and biological processes of the cell are at the minimum. Therefore, the efficacy and applicability of any phlorotannin derivative will be dependent on the safety-to-toxicity curve ratio, especially when related to human and in vivo trials (Barbosa et al. 2019). As concluded from the limited number of studies on toxicity and safety of phlorotannins, dieckol, was generally nontoxic with rarely mild detected adverse effects. Other phlorotannins, for example 6, and DPHC (diphloroethohydroxycarmalol) showed no toxicity at low to moderate concentrations on the target cell line in vitro either in lower animals or in invertebrates (Okeke et al. 2021). In a review, Okeke et al. (2021) informed that phlorotannins were of low or nontoxic for a variety of human cell lines (such as Hela and HaCaT), in animal models, in fibroblast cells, and in melanoma cells. Moreover, phlorotannins displayed low toxicity in microalgae, seaweed spores, plants, and humans at a modest dosage, with mildly noticeable contraindications in humans, fish, and dogs (Negara et al. 2021). These findings supported our results and confirmed the promising safety of phlorotannins extracted from P. myrica.

UPLC-MS/MS analysis of phlorotannins extracted from P. myrica brown seaweed
Although quantitative methods of Folin-Ciocalteu and DMBA are easily used to give a general estimation of the phlorotannins amount in an extract, they do not provide data for the qualitative phlorotannin profile. Chromatographic techniques like liquid chromatography-mass spectrometry (LC-MS) and UPLC-MS/MS are neccesary. Up till now, few studies employing these advanced chromatographic methods have achieved considerable characterization of phlorotannins such as , Lopes et al. (2018), and Catarino et al. (20192022), and Chouh et al. (2022) Twenty seven different molecular weights of phloroglucinol polymer units ranging from 2 to 8 were recorded for P. myrica extract as listed in Tables 5 and 6. As described by  and Nair et al. (2019), the MS data alone is not usually enough to identify phlorotannins' structure, yet, based upon the acquired tandem MS and the documented literature, a probable chemical structure of these compounds can be depicted. According to Allwood et al. (2020), the negative mode data have more intense signals and allow the detection of molecular weight species of more than 2000 amu (atomic mass unit), which would not have been sensed in the positive mode. In Table 6, peak 3 was detected at a retention time of 0.80 min with a protonated molecular mass at m/z 497.0725, suggesting the presence of phlorotannin tetramer (DP 4).

Conclusion
Recently, a lot of attention has been paid to natural materials to develop a more hygienic human lifestyle, especially nutraceuticals and pharmaceutical products. As established in this study, marine brown seaweed accumulate large amounts of phlorotannin materials. These compounds showed valuable antioxidant properties using several assays. This implies the ability to use these compounds to mitigate oxidative stress cascades that are believed to cause numerous chronic diseases. The effect of phlorotannins was found to be dependent on the species, the concentration, and the chemical structure of each individual extract. In this connection, P. myrica, S. trinodis and T. decurrens as well as D. spiralis phlorotannins extracts, were promising as antioxidant and antidiabetic agents, while S. euryphyllum exhibited valuable anti-inflammatory effects. In addition, phlorotannin extracts were able to completely inhibit acetyl cholinesterase (AChE) activity at high concentrations. However, little is still known about the characterization and structure of phlorotannins to enable their sustainable usage in industrial applications. UPLC-MS/MS revealed that the P. myrica extract encompassed several fuhalol, phlorethol, and fucophlorethol, phlorotannin derivatives in significant ratios with 3-8 units of phloroglucinol. Together with its verified biosafety, this study recommends the phlorotannin extract of P. myrica for antioxidant, antidiabetic, and anti-inflammatory therapeutic applications. Further analytical research will be conducted in the future for phlorotannins from the other investigated seaweeds to enable their possible utilization.
Conflict of interest All authors declared that there is no conflict of interest.
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