Comparative Study on the Effect of Phenolics and Their Antioxidant Potential of Freeze-Dried Australian Beach-Cast Seaweed Species upon Different Extraction Methodologies

Brown seaweed is rich in phenolic compounds and has established health benefits. However, the phenolics present in Australian beach-cast seaweed are still unclear. This study investigated the effect of ultrasonication and conventional methodologies using four different solvents on free and bound phenolics of freeze-dried brown seaweed species obtained from the southeast Australian shoreline. The phenolic content and their antioxidant potential were determined using in vitro assays followed by identification and characterization by LC-ESI-QTOF-MS/MS and quantified by HPLC-PDA. The Cystophora sp. displayed high total phenolic content (TPC) and phlorotannin content (FDA) when extracted using 70% ethanol (ultrasonication method). Cystophora sp., also exhibited strong antioxidant potential in various assays, such as DPPH, ABTS, and FRAP in 70% acetone through ultrasonication. TAC is highly correlated to FRAP, ABTS, and RPA (p < 0.05) in both extraction methodologies. LC-ESI-QTOF-MS/MS analysis identified 94 and 104 compounds in ultrasound and conventional methodologies, respectively. HPLC-PDA quantification showed phenolic acids to be higher for samples extracted using the ultrasonication methodology. Our findings could facilitate the development of nutraceuticals, pharmaceuticals, and functional foods from beach-cast seaweed.


Introduction
Marine biological resources have come under increased attention in the past decade due to their natural bioactive compounds having potential as leads for the development of new functional foods or drugs. Seaweeds, which are part of aquatic ecosystems, have functional properties that make them useful compounds for various industries, including food, cosmeceuticals, bio-stimulant, animal feed, and fertilizer [1,2]. Seaweeds are rich in non-nutritional compounds that are beneficial to human health, such as phenolics compounds [3]. These compounds are secondary metabolites that are produced as part of the seaweed's defence mechanism, via the shikimate/phenylpropanoid pathway [4]. Due to the presence of phenolic compounds, seaweeds can have some ability to improve the immune system, protect against radiation, and contribute to the treatment of chronic diseases, including cardiovascular disease, obesity, cancer, and diabetes, as well as neuroprotective diseases including epilepsy, Parkinson's, autism, and Alzheimer's [5].
In Australia, large amounts of seaweed biomass accumulate on the shores due to storms, winds, and currents, which results in seaweed becoming detached and washed Table 1. Estimation of freeze-dried total phenolics of seaweed species extracted by the conventional and non-conventional method of ultrasonication.  The total phenolic content (TPC) among the seaweed species revealed a significant difference (p < 0.05), according to the Tukey statistical analysis (Table 1). Further data analysis indicates ultrasonication to be a more effective methodology for extracting total phenolic content (TPC) than the conventional method. Among the species, Cystophora sp. showed significant TPC values (p < 0.05) when extracted with 70% acetone (19.35 mg GAE/g, ultrasonication; 18.27 mg GAE/g, conventional), 70% ethanol (28.92 mg GAE/g, ultrasonication; 15.49 mg GAE/g, conventional), and 70% methanol (22.74 mg GAE/g, ultrasonication; 18.59 mg GAE/g, conventional), while negligible amounts were found in absolute ethyl acetate (1.66 mg GAE/g, ultrasonication; 0.96 mg GAE/g, conventional). In ultrasonication extracts, phenolic content ranged from 0.34 mg GAE/g (Sargassum sp., absolute ethyl acetate) to 28.92 mg GAE/g (Cystophora sp., 70% ethanol), whereas in conventional extraction, phenolic content ranged from 0.09 mg GAE/g (Durvillaea sp., absolute ethyl acetate) to 18.59 mg GAE/g (Cystophora sp., 70% methanol). The higher phenolic content from ultrasonication might be due to the ability to break the cell walls and facilitate extraction of phenolic compounds [22]. Previously, Carpophyllum plumosum (sub-tropical region, New Zealand) was identified to have significantly higher values (p < 0.05) than Phyllospora comosa (temperate region, Tasmania) using conventional methodology, which is consistent with our study [23]. In another study, the Australian seaweeds collected from Bateau Bay demonstrated the total phenolic content of Sargassum vestitum (141.91 mg GAE/g), Sargassum linearifolium (47.06 mg GAE/g), Phyllospora comosa (67.78 mg GAE/g), and Sargassum podacanthum (43.13 mg GAE/g) extracted using the ultrasonication method with 70% ethanol [24]. The variation in phenolic content could be due to differences in seaweed species and sampling locations [25]. According to our study, ultrasonication extraction generally produces higher TPC values compared to conventional extraction. The 70% ethanol is the most effective extracting solvent for most seaweed samples, especially when used with ultrasonication extraction. The 70% methanol and 70% acetone produce moderate TPC values, regardless of the extraction methodologies used. Absolute ethyl acetate produced the lowest TPC values for all seaweed samples, regardless of the extraction methods used.
Flavonoids are a widespread and diverse group of natural compounds. These compounds possess biological activities including radical scavenging activity [26]. The total flavonoids content of the seaweed samples was determined using the aluminium chloride method. A significant difference (p < 0.05) in the total flavonoid content was observed among the extraction, species, and solvents in our study. The total flavonoid content ranged between 0.67 mg QE/g (Sargassum sp., 70% acetone) to 18.23 mg QE/g (Durvillaea sp., absolute ethyl acetate) in ultrasonication whereas the conventional extraction ranged from 0.04 mg QE/g (Durvillaea sp., 70% methanol) to 6.52 mg QE/g (Phyllospora comosa, absolute ethyl acetate). The total flavonoid content was high in absolute ethyl acetate extracted by the conventional methodology of Phyllospora comosa (6.52 mg QE/g), Durvillaea sp. (6.35 mg QE/g) and Sargassum sp. (6.35 mg QE/g) whereas the ultrasound extraction of absolute ethyl acetate (Durvillaea sp., 18.23 mg QE/g) and 70% methanol solvent (Ecklonia radiata, 11.15 mg QE/g) exhibited higher flavonoid content. The findings of this investigation reveal that ultrasound extraction resulted in higher flavonoid concentration, possibly partly due to degradation of the flavonoid compound during long processing times during conventional methodology [27]. Our study estimated the flavonoid content extracted by the solvent 70% ethanol of Sargassum sp., to be 2.79 mg QE/g (ultrasonication) and 0.96 mg QE/g (conventional) whereas another seaweed reported the ethanol extracted from Sargassum sp., to be 42.6 mg QE/g [28]. The difference in the flavonoid content is likely due to the different collection regions of the Sargassum sp.
The extraction of bioactive compounds from seaweed is an important step in the development of functional foods and nutraceuticals with potential health benefits. The efficiency of extraction methods can vary depending on the sample and the solvent used. The results of this study suggest that ultrasonication is generally a more efficient method of extraction for obtaining higher TPC, TFC, TCT, DMBA, PBA, and FDA values compared to conventional extraction methods. The choice of extraction solvent can also have a significant impact on the chemical composition of each sample.

Antioxidant Potential
Seaweeds have been widely studied for their antioxidative properties due to the presence of phenolic compounds [31]. The study utilised a diverse range of methods, including the DPPH, ABTS, FRAP, FICA, OH-RSA, RPA, and TAC assays, to measure the antioxidant activity in various extracts obtained from five different species of seaweed. In this study, antioxidant potential are shown in Table 2, which is combined data for free and bound (Tables S3 and S4) phenolics in the seaweed samples. The data was analysed using the Tukey statistical analysis method to compare the antioxidant activity of the different extracts. Seaweed is a complex system, and therefore, using several antioxidant assays is necessary to account for the different interactions present. There is no single method that can accurately measure the antioxidant properties of seaweed. Thus, using a combination of assays provides a more comprehensive understanding of the antioxidant capabilities of seaweed [32].
One of the most commonly used methods to evaluate antioxidant activity is the DPPH method, which is widely recognised for its effectiveness in assessing the antioxidant potential of various samples. Table 2 shows the DPPH scavenging capacities of five different seaweed species using various solvents and extraction methods. The study found significant variations in DPPH scavenging activity among the species, methods, and solvents used. The conventional extraction method showed similar antioxidant potential as ultrasonication. The highest scavenging activity was found in the 70% acetone extract of Cystophora sp. followed by Sargassum sp., Phyllospora comosa, Ecklonia radiata, and Durvillaea sp. when using ultrasonication. On the other hand, the highest scavenging activity was found in the 70% acetone extract of Cystophora sp., followed by Ecklonia radiata, Phyllospora comosa, Sargassum sp., and Durvillaea sp., when using conventional extraction. Although acetone was found to be the most effective solvent for extraction antioxidants in our study, previous research has demonstrated high antioxidant activity in Ecklonia sp. using ethanol solvent and ultrasonication extraction [33]. It is important to note that there are no ideal organic solvents that would extract total antioxidants, as phenolics can vary in polarity and also be bound with carbohydrate or protein [34]. Additionally, a previous study has demonstrated the presence of DPPH stable free radical scavenging activity in Sargassum sp., which supports the findings of our study [35].
FRAP is an important antioxidant assay used to measure the reducing power of a sample. Table 2 summarises the antioxidant capacities of different seaweeds, which were extracted via conventional and ultrasound methods. Among the species analysed, the 70% acetone extract of Cystophora sp. had the highest FRAP value, followed by 70% acetone extract of Sargassum sp. and 70% methanol extract of Sargassum sp., both obtained from ultrasound extraction. On the other hand, Cystophora sp. (33.48 mg TE/g, 70% methanol; 33.45 mg TE/g, 70% ethanol; 32.54 mg TE/g, 70% acetone) had higher antioxidant potential than other species using conventional methodology. Overall, Cystophora sp., Sargassum sp., and Ecklonia radiata had higher antioxidant potential in solvents of 70% methanol, 70% ethanol, and 70% acetone. However, negligible antioxidant potential was detected in the solvent of absolute ethyl acetate. Our study demonstrated lower antioxidant potential in 70% ethanol extracted Durvillaea sp. (11.39 mg TE/g, ultrasonication; 5.83 mg TE/g; conventional) when compared with another study that showed the antioxidant potential of Durvillaea antarctica extracted with absolute ethanol to be 20.6 mg TE/100 g and 50% ethanol to be 80.3 mg TE/100 g [36]. The reason might be due to difference in species, the location of collection of the seaweed, and environmental conditions [25].
ABTS is a commonly used method for measuring the antioxidant activity of a substance. It measures the ability of an antioxidant to scavenge ABTS radical cations and convert them into a colorless form [37]. In the current study, significant differences (p < 0.05) were observed among seaweed species in the ABTS assay. The antioxidant potential ranged from 4.86 mg TE/g (Sargassum sp., ethyl acetate) to 64.15 mg TE/g (Cystophora sp., 70% acetone) in the ultrasonication method. In contrast, the antioxidant potential ranged from 1.87 mg TE/g (Durvillaea sp., ethyl acetate) to 42.5 mg TE/g (Cystophora sp., 70% acetone) using the conventional methodology. The antioxidant potential of Cystophora sp. was high in both extraction methodologies and in solvents of 70% acetone, 70% methanol, and 70% ethanol. However, a previous study showed that Durvillaea antarctica extracts were 0.71 mg TE/100 g (absolute ethanol) and 6.34 mg TE/100 g (50% ethanol) [36] while our study showed higher antioxidant potential in 70% ethanol extract (18.36 mg TE/100 g, ultrasonication; 12.29 mg TE/100 g, conventional). The difference in results might be due to difference in varieties, growing region, extraction solvent, and solute to solvent ratio.  In ·OH-RSA, the ability of seaweed samples to scavenge hydroxyl radicals were measured [38]. Our study found that the ultrasonication method was more effective than the conventional methodology in extracting antioxidants. Among the seaweeds analysed, Ecklonia radiata (134.49 mg TE/g, 70% acetone) had the highest antioxidant potential using ultrasonication, followed by Phyllospora comosa (65.42 mg TE/g, 70% methanol) and Cystophora sp. (62.67 mg TE/g, 70% methanol). However, the 70% methanol extract of Phyllospora comosa (26.63 mg TE/g) and the 70% acetone extract of Sargassum sp. (23.37 mg TE/g) had higher antioxidant potential than Durvillaea sp. (16.80 mg TE/g, 70% methanol), Ecklonia radiata (6.75 mg TE/g, 70% methanol), and Durvillaea sp. (6.50 mg TE/g, 70% ethanol), when using the conventional methodology. It is worth noting that no antioxidant activity was detected when using absolute ethyl acetate solvent in conventional extraction. Overall, the ultrasonication method exhibited higher antioxidant activity compared to other methods. Among the solvents tested, 70% methanol and 70% acetone were found to have the highest antioxidant potential.
In the FICA assay, the chelating ability of seaweed samples was measured by the conversion of ferrozine to ferrous ions [39]. In FICA, among the extractions carried out using ultrasonication, the 70% acetone extract of Phyllospora comosa showed the highest antioxidant potential (4.14 mg EDTA/g). In the case of conventional extraction, the best antioxidant potential was found in the absolute ethyl acetate extract of Ecklonia radiata (4.46 mg EDTA/g, ethyl acetate). According to the results of Table 2, ultrasonication generally showed higher values of antioxidant potential than the conventional method.
Total Antioxidant Capacity (TAC) is a measure of the ability of a substance to neutralise free radicals and protect against oxidative stress. TAC is often used as an indicator of the antioxidant potential of foods and natural products, such as seaweed. In TAC, the different species of seaweed are considered to have high bioactive compounds such as total phenolics and flavonoid content, which contribute to their total antioxidant activity. The seaweed with the highest TAC was Cystophora sp., extracted using ultrasonication method with 70% acetone extraction, followed by Sargassum sp. (70% acetone), Phyllospora comosa (70% acetone), Sargassum sp. (absolute ethyl acetate), and Ecklonia radiata (70% acetone). Comparably high TAC was found in Cystophora sp. extracted by conventional method of 70% acetone followed by Cystophora sp. (70% ethanol), Sargassum sp. (70% acetone), Ecklonia radiata (70% ethanol), Ecklonia radiata (70% methanol), and Ecklonia radiata (70% acetone). The solvents that extracted the highest antioxidant potential within the species were 70% acetone, 70% ethanol, and 70% methanol.

Correlation
Pearson's correlation analysis was performed to observe the correlations between phenolic compounds and antioxidant potential for both ultrasonication and conventional methodologies (Table 3). In ultrasonication extraction, TPC showed a significant correlation (p < 0.05) with FDA, PBA, DPPH, FRAP, ABTS, TAC, and RPA. The correlation between TFC and DMBA is significant (p < 0.05) in both conventional and ultrasonication methodologies. The ultrasonication methodology has shown moderate to strong positive correlations between DPPH and FRAP (r = 0.657), ABTS (r = 0.690), OH-RSA (r = 0.474), TAC (r = 0.646), and RPA (r = 0.480). However, FICA only displayed a weak positive correlation (r = 0.406) with DPPH. Stronger positive correlations were found between DPPH and FRAP (r = 0.824), ABTS (r = 0.809), TAC (r = 0.809), and RPA (r = 0.688) using the conventional extraction method. It is worth noting that FICA displayed a negative correlation (r = −0.376) with DPPH in conventional extraction. Similarly, through conventional extraction, TPC exhibited a high correlation with DPPH, FRAP, ABTS, PBA, and FDA. TFC showed a significant correlation with DMBA (p < 0.05), which was similar to ultrasonication. In both conventional and ultrasonication methodologies, FRAP is significantly correlated with ABTS, RPA, and TAC. Previous studies have shown significant correlation between TPC and DPPH in Acanthophora spicifera (Rhodophyta) [40]. In another seaweed study, gallic acid has been found to have a positive correlation with DPPH scavenging activity [41]. Matanjun et al. [42] reported that ABTS had strong correlation with FRAP, which supports our study. Table 3. Pearson's correlation coefficients (r) of phenolic contents and the antioxidant capacity for ultrasonication and conventional methodologies. In addition, principal components analysis (PCA, Figure 1) was performed to investigate the overall relationship between the phenolic content and their antioxidant potential extracted via the methodology of conventional and ultrasonication. In ultrasonication, TPC is highly correlated with ABTS, DPPH, TAC, FRAP, and RPA which is same as Pearson's correlation coefficients (r). In conventional methodology, the TPC is highly correlated with PBA, FDA, DPPH, FRAP, ABTS, OH-RSA, and RPA, similar to Pearson's correlation coefficients (r). Pharmaceuticals 2023, 16, x FOR PEER REVIEW 11 of 41

LC-ESI-QTOF-MS/MS Characterization of Phenolic Compounds
LCMS/MS has been widely used in the identification and characterization of the phenolic compound present in the marine seaweed [52]. Qualitative analysis of the phenolic compounds was performed via LC-ESI-QTOF-MS/MS in both positive and negative modes of ionization. Compounds with mass error < ±5 ppm and PCDL library score more than 80 were selected for further MS/MS identification and m/z characterization purposes. In the present work, the MS/MS was performed and 94 and 104 compounds were identified in extracts from ultrasonication and conventional methodology, respectively (Tables 4 and 5), which is combined data for both free and bound phenolic compounds (Tables S5  and S6). The phenolic acids present in extracts from ultrasound and conventional

LC-ESI-QTOF-MS/MS Characterization of Phenolic Compounds
LCMS/MS has been widely used in the identification and characterization of the phenolic compound present in the marine seaweed [52]. Qualitative analysis of the phenolic compounds was performed via LC-ESI-QTOF-MS/MS in both positive and negative modes of ionization. Compounds with mass error < ±5 ppm and PCDL library score more than 80 were selected for further MS/MS identification and m/z characterization purposes. In the present work, the MS/MS was performed and 94 and 104 compounds were identified in extracts from ultrasonication and conventional methodology, respectively (Tables 4 and 5), which is combined data for both free and bound phenolic compounds (Tables S5 and S6). The phenolic acids present in extracts from ultrasound and conventional methodology were 34 and 33 compounds, respectively. The flavonoids were 43 and 52 in ultrasonication and conventional, respectively. The anthocyanins, a sub-class of flavonoids, were only observed in conventional extraction methods. In other polyphenols, 17 and 19 compounds were identified after MS 2 in ultrasonication and conventional, respectively.                  [53,54]. Biosynthesis of gallic acid is formed from 3-dehydroshikimate in the presence of shikimate dehydrogenase enzyme to produce 3,5-didehydroshikimate. Further, the 3,5-didehydroshikimate compound rearranges the structure spontaneously to form gallic acid [55]. In the ultrasonication method, Cystophora sp. (bound phenolics of ethyl acetate extract) detected the presence of 2,3-dihydroxybenzoic acid while for gallic acid was identified in free and bound forms of phenolics in Phyllospora comosa (70% acetone, 70% ethanol, 70% methanol extract), Ecklonia radiata (70% acetone, 70% ethanol, absolute ethyl acetate, 70% methanol extract), Sargassum sp. (absolute ethyl acetate extract), and Cystophora sp. (absolute ethyl acetate, 70% ethanol, 70% acetone extract), whereas 2-hydroxybenzoic acid was present in bound phenolics of Sargassum sp. (acetone, methanol extract). In conventional methodology, Ecklonia radiata detected the compounds 2-hydroxybenzoic acid and 2,3-dihydroxybenzoic acid in bound phenolics, while gallic acid was identified in Phyllospora comosa, Ecklonia radiata, Durvillaea sp., and Sargassum sp. in free form of phenolics. However, Phyllospora comosa (70% acetone extract) and Sargassum sp. (70% ethanol extract) detected gallic acid in bound form as well. Previously, gallic acid was detected in Himanthalia elongata (Phaeophyceae) and Ulva intestinalis (Chlorophyta) [56,57]. Seaweeds including Gracilaria birdiae and Gracilaria cornea (Rhodophyta) collected along the Brazilian shorelines detected the presence of gallic acid. Gallic acid was also detected in other plants including green teas, bearberry leaves, hazelnuts, evening primrose grape seeds [58], and fruit pulp of Terminalia chebula [59]. Gallic acid is known for its anticancer, anti-inflammatory, anti-melanogenic, and antioxidant properties [60]. The 2-hydroxybenzoic acid was previously detected from lucerne, hops, berries, Keitt and Kensington Pride mangoes [61]. The 2-hydroxybenzoic acid is a key ingredient in the skin care industry and is used to treat psoriasis, keratosis pilaris, acne, corns, calluses, and warts [62]. The 2,3-dihydroxybenzoic acid was previously detected in Catharanthus roseus, wild jujube fruit, wild olive fruit, wild common fig fruit, apple, grapes, kiwi fruit, nectarine, peach, orange, pineapple, plum, and passionfruit peels [63].
Ferulic acid was tentatively identified by precursor ions [M -H] − m/z at 193.0516. The compound was confirmed by product ions at m/z 178, m/z 149, and m/z 134, indicating the loss of CH 3 , CO 2 , and CH 3 with CO 2 from the precursor ions, respectively [64]. Ferulic acid is an abundant hydroxycinnamic acid, available in free form but linked to the lignin. It acts as a precursor in the plant defence response for the production of phytoalexins, signaling molecules and antimicrobial compounds [65]. In our study, ferulic acid was identified in free form from 70% acetone ultrasonic extract of Cystophora sp. Previously, ferulic acid has been detected in some seaweed, including Bifurcaria bifurcate, Ascophyllum nodosum, and Fucus vesiculosus (Phaeophyceae) [66]. Another study detected that Himanthalia elongata collected from Ireland contained ferulic acid [67]. Similarly, seeds of coffee, artichoke, peanuts, bamboo shoots, eggplant, soybean, spinach, tomato, radish, broccoli, carrot, avocado, orange, banana, berries, and coffee [68] contain ferulic acid. Ferulic acid exhibits antioxidant, anti-inflammatory, antimicrobial, anti-allergic, anti-thrombosis, and anti-cancer activities [69]. m-Coumaric acid ([M -H] − m/z at 163.0412), was identified at product ions m/z 119, due to the loss of CO 2 (44 Da) [64]. In ultrasonication methodology, the compound was detected in bound form in Durvillaea sp. (70% acetone, 70% ethanol, 70% methanol), Sargassum sp. (70% acetone), and Cystophora sp. (70% acetone), whereas in conventional methodology, Cystophora sp. (absolute ethyl acetate) only identified the compound in free form. Coumaric acid is present in various berries such as strawberries, peanuts, beers, olive oil, and baru almonds [70]. m-Coumaric acid compound have antioxidant capacity [71]. Previously, a study on m-coumaric acid reported that the compound reduced glucose and glycated hemoglobin levels and enhanced antioxidant activity [72].  [75]. However, the compound myricetin 3-O-arabinoside was detected in American cranberry and highbush blueberry [76] in very limited studies on their biological properties. The compound quercetin was also identified and characterised in Durvillaea sp. [77].
Resveratrol  [87]. It was identified in the seaweeds Sargassum sp. (70% acetone, 70% ethanol) and Cystophora sp. (70% acetone) in ultrasonication. The compound was previously detected in red wine and contributes to the antioxidant potential and hence may play a role in the prevention of cardiovascular diseases [88].

Heatmap Analysis of Quantified Phenolics in Seaweeds
Quantification of phenolic compounds in seaweed has been a topic of research and discussion for many years. In this study, the phenolic compounds were quantified using high performance liquid chromatography connected to photodiode array detector (HPLC-PDA). This method quantifies individual compounds based on their retention time and UV absorption spectra. HPLC-PDA is very specific and accurate when compared to the in vitro assay estimation completed earlier in this study. The heat map ( Figure 6A,B) was constructed based on the data of the combined results of both free and bound phenolics in Australian brown beach-cast seaweeds extracted via conventional and ultrasonication methodologies. In this study, twelve phenolics were quantified, including ten phenolic acids and two flavonoids. Phloroglucinol extracted via ultrasonication extraction was quantified and shown to be present in high amounts in Cytosphora sp. (70% acetone extract), and Sargassum sp. (70% acetone extract). However, in conventional extraction, the compound was more abundant in Sargassum sp. (70% acetone extract), followed by Ecklonia radiata (70% ethanol extract), but lower than obtained using ultrasonication extraction. The differences in phlorotannin levels are probably influenced by species, season, and the site of collection [89]. A study demonstrated that 70% acetone was the highest extracted level of phenolics from the seaweeds, which is consistent with our study [90]. In seaweeds, gallic acid is metabolised via dehydrogenation of 5-dehydroshikimic acid [91]. In our study, we quantified gallic acid and observed that ultrasonication extraction extracted a higher amount of the compound than conventional extraction. This might be due to increase in mass transfer in reduced extraction time in ultrasonication [12]. Pyrogallol was only identified and quantified in Phyllosphora Comosa (70% acetone extract) via the ultrasonication methodology, whereas this compound was identified and quantified in Ecklonia radiata, Durvillaea sp., Sargassum sp., and Cytosphora sp., extracted via the conventional methodologies. Ultrasonic extraction might partially degrade some targeted compounds due to shear force and temperature, resulting in lower yields for some compounds [92]. Therefore, the conventional extraction may be better for the recovery of compounds sensitive to the higher temperatures whereas shear stress is generated in ultrasonic extraction which results in low recovery of phenolic compounds. Protocatechuic acid is formed from the dehydration of the 3-dehydroshikimic acid in the metabolic pathway of the seaweeds [93]. The compound was higher in ultrasonication of the Sargassum sp. (70% acetone extract) when compared to other species and solvents. However, protocatechuic acid compound was not detected in the conventional extraction, illustrating an example where ultrasonic extraction is more effective.

Seaweed Collection and Identification of Seaweed Samples
Phyllospora comosa, Ecklonia radiata, Durvillaea sp., Sargassum sp., and Cystophora sp. (Phaeophyceae) seaweeds ( Figure 7) were abundantly found at Queenscliff Harbour (38°15′54.0″ S 144°40′10.3″ E), Victoria, Australia and collected in the month of February (summer). A randomised collection pattern was used without considering the age and size of the seaweed. These seaweed samples were identified at Deakin Marine Institute, Queenscliff, Victoria, Australia.

Sample Preparation
Fresh seaweed samples were thoroughly washed with tap water and subsequently with Milli-Q water to remove any external adhering salts, epiphytes, and other foreign impurities. The seaweed samples were cut manually into smaller pieces about 1-3 cm each with a stainless-steel food-grade knife. The fresh seaweed samples were freeze-dried. The seaweed samples were frozen to −70 °C for 24 h in a Thermo scientific freezer. The frozen samples were placed in the freezer dryer at −60 °C for 72 h as the procedure described in Badmus et al. [94]. The sample was ground using a grinder (Cuisinart Nut and Spice

Sample Preparation
Fresh seaweed samples were thoroughly washed with tap water and subsequently with Milli-Q water to remove any external adhering salts, epiphytes, and other foreign impurities. The seaweed samples were cut manually into smaller pieces about 1-3 cm each with a stainless-steel food-grade knife. The fresh seaweed samples were freeze-dried. The seaweed samples were frozen to −70 • C for 24 h in a Thermo scientific freezer. The frozen samples were placed in the freezer dryer at −60 • C for 72 h as the procedure described in Badmus et al. [94]. The sample was ground using a grinder (Cuisinart Nut and Spice grinder 46302, Melbourne, VIC) to make it into a fine coarse powder. The dried samples were stored in the cold room.

Free Phenolics Extraction
The conventional and ultrasonication of free phenolics were performed and slightly modified as described byČagalj et al. [95]. The seaweed samples, in triplicate, were extracted with 70% methanol, 70% ethanol, 70% acetone, and absolute ethyl acetate using two extraction methods. All the extraction solvents were added with 0.1% formic acid. The seaweed to solvent ratio was set at 1:20 for all extractions. The following extraction methods were applied: (i) shaking incubator for 16 h at 120 rpm at 10 • C (ZWYR-240 incubator shaker, Labwit, Ashwood, VIC, Australia) (ii) UAE performed with ultrasonicator at 40% amplitude for 5 min. After the extraction, the samples were centrifuged for 15 min at 5000 rpm under 4 • C using Hettich Refrigerated Centrifuge (ROTINA380R, Tuttlingen, Baden-Württemberg, Germany). The supernatant fluid was filtered via 0.45 µm syringe filter (Thermo Fisher Scientific Inc., Waltham, MA, USA) and collected as free phenolic extracts. The sample residues were air-dried for 72 h. The residues were washed with their solvents 3 times and the residue was then further analysed for bound phenolics.

Bound Phenolic Extraction
The conventional and ultrasonication of bound phenolics were performed and slightly modified as described by Gulsunoglu et al. [96]. The seaweed samples, in triplicate, were extracted with 70% methanol, 70% ethanol, 70% acetone, and absolute ethyl acetate. All solvents were added with 0.1% formic acid. The residue was added with 10 mL 2 N NaOH in a screw-capped test tube. For conventional extraction, the sample was neutralised (pH 7) with 2 N HCl and dosed with 10 mL of respective solvents. The samples were incubated in a shaking incubator for 16 h at 120 rpm at 4 • C (ZWYR-240 incubator shaker, Labwit, Ashwood, VIC, Australia). For ultrasonication, the sample was sonicated at 40% amplitude for 5 min and neutralised (pH 7) with 2 N HCl. Later, the sonicated sample was dosed with

Estimation of Phenolic and Antioxidant Assays
The assays were performed according to the published methods of Subbiah et al. [97] and Suleria, Barrow, and Dunshea [63] for phenolic estimation of free and bound phenolics (TPC, TFC, DMBA, PBA, and FDA) and their total antioxidant potential (DPPH, FRAP, ABTS, ·OH-RSA, FICA, RPA, and TAC) extracted via conventional and ultrasonication methodologies. Multiskan ® Go microplate photometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) were used to attain the absorption data.

Estimation of Total Phenolic Compound
The total phenolic content of free and bound extracted through conventional and ultrasonication methodologies were estimated by Folin-Ciocalteu's method as described in Mussatto et al. [98]. An amount of 25 µL extract, 25 µL Folin-Ciocalteu's reagent solution (1:3 diluted with water) and 200 µL water were added to the 96-well plate (Costar, Corning, NY, USA). The 96-well plate was incubated in the darkroom for 5 min at room temperature (~25 • C). An amount of 25 µL of 10% (w:w) sodium carbonate was added to the reaction mixture and incubated at 25 • C for 60 min. Absorbance was measured at 765 nm using a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Concentrations ranging from 0 to 200 µg/mL of gallic acid were prepared as a standard curve and the TPC content was expressed in mg of gallic acid equivalents per gram based on dry weight (mg GAE/g of the sample).

Determination of Total Flavonoid Compounds (TFC)
The quantification of TFC was completed by using the aluminum chloride method with slight modification as described in Ali et al. [99]. The extract of the phenolic compounds were extracted through conventional and ultrasonication methodologies. An amount of 80 µL extract followed by 80 µL of aluminum chloride and 120 µL of 50 g/L sodium acetate solution were added to the 96-well plate. The 96-well plate was incubated for 2.5 h in the darkroom. Absorbance was measured at 440 nm. The concentration ranging from 0-50 µg/mL for the quercetin calibration curve was used to determine TFC and expressed in mg quercetin equivalents per gram of sample (mg QE/g d.w. ).

Determination of Total Tannin Content (TTC)
The total tannin content was determined by the vanillin sulfuric acid method as described by Ali, Wu, Ponnampalam, Cottrell, Dunshea and Suleria [99] with slight modification. As previously mentioned, the phenolic compounds were extracted through conventional and ultrasonication methodologies. An amount of 25 µL of sample extract followed by 25 µL of 32% sulfuric acid and 150 µL of 4% vanillin solution was added to a 96-well plate and incubated for 15 min in the darkroom. The absorbance was measured at 500 nm. Catechin calibration curve with concentration from 0 to 1 mg/mL was used for estimation of TCT and expressed in mg catechin equivalents (CE) per g of sample weight (mg CE/g d.w.).

2,4-Dimethoxybenzaldehyde Assay (DMBA)
The total phlorotannin content was estimated using the 2,4-dimethoxybenzaldehyde (DMBA) assay as described in Vissers et al. [100]. An amount of 2% of DMBA was added in acetic acid (m/v) and 6% hydrochloric acid in acetic acid (v/v). Both solutions were mixed at equal volumes to make DMBA solution. An amount of 25 µL of sample and 125 µL of DMBA solution were added into the 96-well microplate. The reaction mixture was incubated in the dark at 25 • C for 60 min. The absorbance was read at 510 nm. The standard curve was prepared to estimate total phlorotannin of phloroglucinol (0-25 µg/mL) and expressed in mg phloroglucinol equivalents per gram (mg PGE/g d.w ).

Prussian Blue Assay (PBA)
To estimate the total phlorotannin content, the methodology was first suggested by Stern et al. [101] and modified according to Margraf et al. [102]. A diluted sample of 50 µL was added to 50 µL of ferric ammonium sulfate (0.1 M FeNH 4 (SO4) 2 in 0.1 M HCl) and the reaction mixture was kept in dark for 2 min and an addition of 50 µL of potassium ferricyanide [0.008 M K 3 Fe(CN) 6 ]. It was incubated in a dark room for 15 min and absorbance recorded at 725 nm. The standard curve was prepared to estimate total phlorotannin of phloroglucinol (0-3.125 µg/mL) and expressed in mg phloroglucinol equivalents per gram (mg PGE/g d.w ).
3.5.6. Folin-Denis Assay (FDA) Phlorotannin assay was determined by Folin-Denis assay as described in Stern, Hagerman, Steinberg, Winter and Estes [101]. The reagent of Folin-Denis was prepared by dissolving 25 g of sodium tungstate (Na 2 WO 4 ·2H 2 O) and 5 g of dodeca-molybdophosphoric acid (12MoO 3 ·H 3 PO 4 ·H 2 O) in 175 mL distilled water, adding 12.5 mL phosphoric acid to the solution, boiling under reflux for 2 h, and then makeup to 250 mL. An amount of 5 µL of the sample was mixed with 20 µL of Folin-Denis reagent, 40 µL of saturated sodium carbonate, and 125 µL of water. The reaction mixture was incubated in the dark for 2 h and the absorbance was read at 725 nm. The standard curve estimates the total phlorotannin of phloroglucinol (0-100 µg/mL) and is expressed in mg phloroglucinol equivalents per gram (mg PGE/g d.w ).

2,2 -Diphenyl-1-Picrylhydrazyl (DPPH) Assay
The estimation of free radical scavenging activity of the seaweed by the DPPH method was performed as described by Nebesny and Budryn [103] with slight modification. To prepare the DPPH radical solution, dissolve 4 mg of DPPH in 100 mL of analytical-grade methanol. An amount of 40 µL of extract and 260 µL of DPPH solution were added to a 96-well plate and vigorously shaken in the dark for 30 min at 25 • C. The absorbance was measured at 517 nm. Trolox standard curve with a concentration ranging from 0 to 200 µg/mL was used to determine the DPPH radical scavenging activity and expressed in mg of Trolox equivalent per gram (mg TE/g d.w. ) of the sample.

Ferric Reducing Antioxidant Power (FRAP) Assay
This assay has been used to estimate the antioxidant capacity in marine seaweeds with some modifications as described by Benzie and Strain [104]. To prepare the FRAP dye, 20 mM Fe [III] solution, 10 mM TPTZ solution, and 300 mM sodium acetate solution were mixed at a ratio of 1:1:10. An amount of 20 µL of the extract and 280 µL prepared dye were added to a 96-well plate and incubated at 37 • C for 10 min. The absorbance was measured at 593 nm. Trolox standard curve with concentration ranging from 0 to 100 µg/mL was used to determine the FRAP values and expressed in mg of Trolox equivalent per gram of sample (mg TE/g d.w .).
3.5.9. 2,2 -Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid (ABTS) Assay ABTS radical cation decolorization assay was used to determine the free radical scavenging activity of the marine seaweed samples with few modifications as described in Re et al. [105]. An amount of 88 µL of 140 mM potassium persulfate and 5 mL of 7 mM ABTS solution were added to prepare the ABTS + stock solution and were incubated in the darkroom for 16 h. An amount of 10 µL of the extract and 290 µL dye solution were added to the 96-well plate and incubated at 25 • C for 6 min. The absorbance was measured at 734 nm. The antioxidant potential was calculated using the standard curve of Trolox with (0-500 µg/mL) and was expressed in Trolox (TE) in mg per gram of sample.

Estimation of Hydroxyl Radical Scavenging Activity (OH-RSA)
The hydroxyl radical scavenging activity of marine seaweed was determined with the modification of the method Smirnoff and Cumbes [106]. An amount of 50 µL sample extract, 50 µL 6 mM hydrogen peroxide, and 50 µL 6 mM ferrous sulfate heptahydrate were injected into the plate and incubated at 25 • C for 10 min. To the reaction mixture, 50 µL of 6 mM 3-hydroxybenzoic acid was added. Trolox (0-400 µg/mL) was used for calibration and the absorbance was measured at 510 nm.
3.5.11. Estimation of Ferrous Ion Chelating Activity (FICA) FICA assay was performed as described by Dinis et al. [107] with slight modifications. An amount of 15 µL sample extract, 85 µL water, 50 µL 2 mM ferrous chloride, and 50 µL of 5 mM ferrozine were added to a 96-well plate. The reaction mixture was incubated for 10 min in the dark at 25 • C. The standard curve of EDTA (0-50 µg/mL) was prepared and the absorbance was measured at 562 nm. The results were expressed as mg EDTA equivalents per dry weight (mg EDTA/g d.w ).

Estimation of Reducing Power (RPA)
The RPA assay was modified and performed according to the method of Ferreira et al. [108]. An amount of 10 µL sample extract, 25 µL 1% potassium ferricyanide (III) solution, and 25 µL 0.2 M phosphate buffer (pH 6.6) were added to a 96-well plate. The reaction mixture was incubated for 20 min at 25 • C. To the reaction mixture, 25 µL of 10% trichloroacetic acid was added to stop the reaction followed by the addition of 85 µL water and 8.5 µL 0.1% ferric chloride solution. It was incubated for 15 min at 25 • C. A standard curve of Trolox (0-500 µg/mL) was used for the calibration curve and absorbance was measured at 750 nm and the results were expressed as mg TE/g ± SD.

Total Antioxidant Capacity (TAC)
The total antioxidant capacity was estimated by the phosphomolybdate method as described in Prieto et al. [109]. An amount of 0.028 M sodium phosphate, sulphuric acid (0.6 M), and 0.004 M ammonium molybdate were mixed to form phosphomolybdate reagent. An amount of 40 µL extract and 260 µL of phosphomolybdate reagent were added to the 96-well plate. The reaction mixture was incubated at 90 • C for 90 min. The absorbance was measured at 695 nm upon the reaction mixture cooling down to room temperature. TAC was determined by using the Trolox standard curve (0-200 µg/mL) and expressed in mg Trolox equivalents (TE) per g of the dry sample weight.

Characterization of Phenolic Compounds by LC-ESI-QTOF-MS/MS Analysis
LC-ESI-QTOF-MS/MS carried out the extensive characterization of phenolic compounds using the method described by Allwood et al. [110] and Zhu et al. [111]. The phenolic compounds from five different species of Australian beach-cast seaweeds were extracted via conventional and ultrasonication methodologies. An Agilent 1200 series of HPLC (Agilent Technologies, Santa Clara, CA, USA) connected via electrospray ionization source (ESI) to the Agilent 6530 Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) LC/MS (Agilent Technologies, Santa Clara, CA, USA). HPLC buffers were sonicated using a 5 L Digital Ultrasonic water bath (Power sonic 505, Gyeonggi-do, Republic of Korea) for 10 min at 25 • C. The separation was carried out using a Synergi Hydro-Reverse Phase 80 Å, LC column 250 × 4.6 mm, 4 µm (Phenomenex, Torrance, CA, 202 USA) with temperature 25 • C and sample temperature at 10 • C. The sample injected was 20 µL. Since the system was binary solvent: mobile phase A, 100% MilliQ water added with 0.1% formic acid, and mobile phase B, acetonitrile/MilliQ water/formic Acid (95:5:0.1), at a flow rate of 0.3 mL/min. The gradient was as follows: 0-2 min hold 2% B, 2-5 min 2-5% B, 5-25 min 5-45% B; 25-26 min 45-100% B, 26-29 min hold 100% B, 29-30 min 100-2% B, 30-35 min hold 2% B for HPLC equilibration. Both positive and negative modes were applied for peak identification. Nitrogen gas has been used as a nebulizer and drying gas at 45 psi, with a flow rate of 5 L/min at 300 • C. Capillary and nozzle voltage were placed at 3.5 kV and 500 V, respectively, and the mass spectra were obtained at the range of 50-1300 amu. Further, MS/MS analyses were carried out in automatic mode with collision energy (10, 15, and 30 eV) for fragmentation. Data acquisition and analyses were performed using Agilent LC-ESI-QTOF-MS/MS Mass Hunter workstation software (Qualitative Analysis, version B.03.01, Agilent).

HPLC-PDA Analysis
The targeted phenolic compounds present in seaweeds were quantified by Agilent 1200 series HPLC (Agilent Technologies, CA, USA) equipped with a photodiode array (PDA) detector according to our previously published protocol of Gu et al. [112] and Suleria, Barrow, and Dunshea [63]. The sample's phenolic compounds were extracted by conventional and ultrasonication. Sample extracts were filtered by the 0. . The flow rate was 0.8, and the column was operated at room temperature. The wavelengths of 280, 320, and 370 nm were simultaneously selected at the PDA detector. Empower Software (2010) was used for instrument control, data collection, and chromatographic processing.

Statistical Analysis
All the analyses were performed in triplicates and the results are presented as mean ± standard deviation (n = 3). The mean differences between different seaweed samples were analysed by one-way analysis of variance (ANOVA) and Tukey's honestly significant differences (HSD) multiple rank test at p ≤ 0.05. ANOVA was carried out via Minitab 19.0 software for windows. For correlations between polyphenol content and antioxidant activities, Pearson's correlation coefficient at p ≤ 0.05 and multivariate statistical analysis including a principal component analysis (PCA), XLSTAT-2019.1.3 were used by Addinsoft Inc., New York, NY, USA.

Conclusions
According to our study, it was found that among the five species of seaweed, Cystophora sp. displayed higher total phenolic and phlorotannin content, as well as antioxidant potential (DPPH, FRAP, ABTS, and TAC). On the other hand, Durvillaea sp. had high flavonoid content but less antioxidant potential. The ultrasonication had extracted higher phenolic and had high antioxidant potential in the solvents of 70% acetone and 70% methanol. The Venn diagram demonstrated high unique compounds in Sargassum sp., and solvent 70% ethanol extracted high unique compounds. In the present work, the MS/MS analysed 94 and 104 compounds in ultrasonication and conventional methodology. The ultrasound and conventional compounds had 72 common compounds whereas the bound and free phenolics had 49 common compounds. The quantification by HPLC showed the quantity of phenolic compounds present, the phenolic acids (phloroglucinol and gallic acid) were higher in ultrasonication when compared to conventional methodology. The findings of the phenolic compounds will further help us in quantifying the compounds and further analysis for the in vitro bio accessibility.