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

Vigasini Subbiah; Faezeh Ebrahimi; Osman T Agar; Frank R Dunshea; Colin J Barrow; Hafiz A R Suleria

doi:10.3390/ph16050773

. 2023 May 22;16(5):773. doi: 10.3390/ph16050773

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

Vigasini Subbiah ^1,², Faezeh Ebrahimi ², Osman T Agar ², Frank R Dunshea ^2,³, Colin J Barrow ¹, Hafiz A R Suleria ^1,^2,^*

Editors: Noelia Duarte, Isabel Ribeiro

PMCID: PMC10224432 PMID: 37242556

Abstract

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.

Keywords: seaweeds, freeze-drying, conventional extraction, ultrasonication, phenolic compounds, antioxidant activity, LC-ESI-QTOF-MS/MS, HPLC-PDA

1. 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 to the shores. The seaweed on the shore can cause strong odour and release greenhouse gases. However, this seaweed could potentially be utilised as a biomass for the production of functional ingredients to develop the latest biotechnological applications [6]. Seaweeds collected from the shore are dried before being used in industrial processing or nutritional evaluation [7], since dried seaweeds can be stored for years without extensive loss of their functional compounds [8]. Researchers have found that freeze-drying is an excellent method for producing high-quality dried products. Cruces et al. [9] reported that freeze drying has the ability to retain high antioxidant potential.

Phenolic compounds exist in free and bound forms. Free phenolics can be extracted easily with solvents, while bound phenolics require various extraction methods, including chemical, biological, and physical methods, as they are entrapped within plant cells [2,10]. The conventional methods used in the extraction are percolation, maceration, heat reflux, and Soxhlet apparatus extractions for extracting phenolic compounds. However, conventional methods can consume large amounts of solvents, produce low yields, and require operating at high temperature that can damage bioactive compounds during extraction [11]. An alternative to conventional methods is ultrasound-assisted extraction, which involves the breakdown of bubbles and disrupts the plant cell walls which thus increases the mass transfer of intracellular components into the solvent [12]. Several studies including Rodrigues et al. [13], Kadam et al. [14] and Hassan, Pham, and Nguyen [15] have used ultrasound extraction in their studies. Ummat et al. [16] conducted a comparative study between ultrasound and conventional extraction of phenolic compounds, phlorotannins, and their antioxidant activities. The ultrasound extraction of the sample produced a higher yield of total phenolics, phlorotannins, flavonoids, and exhibited stronger antioxidant potential.

Various organic solvents can be used for the extraction of phenolic compounds, but the recovery of these compounds depends on the solubility of the phenolics in the solvent. The polarity of the solvent also plays a critical role in enhancing the solubility of phenolics, making it challenging to develop a standardised methodology to extract all phenolics from seaweeds [17]. The evaluation of phenolic compounds can be completed using different spectrophotometric-based in vitro assays [18], including total phenolic content (TPC), total flavonoid content (TFC), total tannin content (TCT), 2,4-dimethoxybenzaldehyde (DMBA), Prussian blue assay (PBA), and Folin–Denis assay (FDA). The antioxidant activity of the phenolic compounds can be assessed using various in vitro methods, such as 2,2′-diphenyl-1-picrylhydrazyl (DPPH) antioxidant assay, 2,2’azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), reducing power assay (RPA), hydroxyl radical scavenging activity (·OH-RSA), ferrous ion chelating activity (FICA), and ferric reducing-antioxidant power (FRAP) assay [19]. Phenolic compounds can be characterised and identified using liquid chromatography coupled with electrospray-ionisation quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS/MS). The compounds are quantified using high performance liquid chromatography (HPLC) combined with photodiode array detector (PDA). According to Zhong et al. [20], the major phenolic compounds identified from eight different seaweed species were gallic acid, cinnamoyl glucose, chlorogenic acid, caffeic acid, and coumaric acid.

In this study, we estimated the total phenolics including free and bound phenolics and their antioxidant potential in freeze-dried samples using two different extraction methodologies that were conventional and ultrasonication extraction, with four different solvents. The phenolics were identified and characterised via LC-ESI-QTOF-MS/MS and the phenolic compounds were quantified using HPLC-PDA. This study has provided information on the phenolic content and antioxidant properties of the freeze-dried seaweeds and thus promotes and provides evidence toward further investigation of their application in food and pharmaceutical industries.

2. Results and Discussion

2.1. Total Phenolics, Flavonoids Tannin and Phlorotannin Content

Bioactive compounds are natural metabolites that are very common in the plant kingdom and provide numerous benefits. Phenolic compounds in particular have attracted great interest in industries ranging from food production to pharmaceuticals due to their protective roles in human health [21]. In this study, total phenolics, flavonoids, tannin and phlorotannin content are shown in Table 1 which combines data for free and bound (Tables S1 and S2) phenolics in the seaweed samples, including Cystophora sp., Phyllospora comosa, Sargassum sp., Ecklonia radiata, Durvillaea sp. There were estimated by ultrasonication assisted extraction (UAE) and conventional extraction using different solvents (70% acetone, 70% methanol, 70% ethanol, and absolute ethyl acetate).

Table 1.

Estimation of freeze-dried total phenolics of seaweed species extracted by the conventional and non-conventional method of ultrasonication.

Samples	Solvents	TPC (mg GAE/g)	TFC (mg QE/g)	TCT (mg CE/g)	DMBA (PGE mg/g)	PBA (PGE mg/g)	FDA (PGE mg/g)
Ultrasonication Extraction
Cystophora sp.	70% ACE	19.35 ± 0.09 ^Ba	2.28 ± 0.02 ^HIb	0.67 ± 0.06 ^EFc	1.96 ± 0.08 ^Fb	2.61 ± 0.30 ^Je	11.21 ± 0.7 ^Ba
	70% MeOH	22.74 ± 0.28 ^Ba	3.33 ± 0.02 ^E–Gb	-	1.02 ± 0.04 ^Ia	8.61 ± 0.08 ^Bb	3.86 ± 0.1 ^Hc
	70% EtOH	28.92 ± 1.33 ^Aa	3.82 ± 0.07 ^DEa	2.21 ± 0.01 ^Ba	1.63 ± 0.02 ^Ga	8.11 ± 0.61 ^BCa	14.2 ± 0.63 ^Aa
	EA	1.66 ± 0.09 ^Ob	7.13 ± 0.9 ^Cb	0.94 ± 0.07 ^DEa	4.15 ± 0.01 ^Dc	5.05 ± 0.25 ^Ha	-
Phyllospora comosa	70% ACE	16.11 ± 0.85 ^Fb	1.86 ± 0.01 ^IJc	0.94 ± 0.07 ^DEb	4.96 ± 0.44 ^Ca	6.18 ± 0.29 ^DEFb	8.57 ± 0.89 ^Cb
	70% MeOH	16.85 ± 0.12 ^Db	3.43 ± 0.06 ^D–Fb	2.86 ± 0.12 ^Aa	0.67 ± 0.01 ^JKb	12.62 ± 0.26 ^Aa	6.58 ± 0.17 ^Ea
	70% EtOH	12.41 ± 0.03 ^Hc	3.62 ± 0.03 ^D-Fb	2.18 ± 0.06 ^Ba	0.46± 0.01 ^LMd	3.2 ± 0.22 ^Ic	6.36 ± 0.41 ^Fb
	EA	2.23 ± 0.04 ^Pd	3.54 ± 0.10 ^D–Fc	0.77 ± 0.06 ^Eb	6.65 ± 0.67 ^Bb	0.13 ± 0.6 ^Lc	-
Sargassum sp.	70% ACE	14.6 ± 0.72 ^Gc	0.67 ± 0.04 ^Le	0.27 ± 0.02 ^Gd	0.89 ± 0.05 ^Id	6.5 ± 0.07 ^DEFa	7.64 ± 0.25 ^Dc
	70% MeOH	16.36 ± 0.34 ^Ec	1.39 ± 0.01 ^JKc	-	0.68 ± 0.04 ^JKb	7.77 ± 0.16 ^Cc	5.24 ± 0.37 ^Gb
	70% EtOH	17.52 ± 0.57 ^Cb	2.79 ± 0.02 ^GHd	0.64 ± 0.03 ^EFc	0.72 ± 0.11 ^Jc	6.85 ± 0.19 ^Db	3.39 ± 0.25 ^Jd
	EA	0.34 ± 0.03 ^Qe	2.6 ± 0.04 ^Hd	-	3.24 ± 0.34 ^Ed	1.18 ± 0.3 ^Kb	-
Ecklonia radiata	70% ACE	11.19 ± 0.41 ^Id	0.85 ± 0.01 ^KLd	1.29 ± 0.01 ^CDa	1.02 ± 0.1 ^Ic	5.86 ± 0.25 ^FGc	2.5 ± 0.04 ^Le
	70% MeOH	12.19 ± 0.41 ^Id	11.15 ± 0.12 ^Ba	0.92 ± 0.09 ^DEc	0.96 ± 0.07 ^a	8 ± 0.37 ^BCc	3.5 ± 0.1 ^IJd
	70% EtOH	9.03 ± 0.11 ^Kd	1.65 ± 0.04 ^Je	-	1.18 ± 0.02 ^Hb	6.13 ± 0.14 ^EFb	0.71 ± 0.01 ^Me
	EA	1.54 ± 0.01 ^Oc	4.04 ± 0.01 ^Dc	-	8.29 ± 0.47 ^Aa	0.96 ± 0.13 ^Kb	-
Durvillaea sp.	70% ACE	9.98 ± 0.68 ^Je	3.35 ± 0.03 ^EFa	1.04 ± 0.01 ^Db	0.57 ± 0.02 ^KLe	5.36 ± 0.14 ^GHd	6.72 ± 0.26 ^Ed
	70% MeOH	4.45 ± 0.09 ^Ne	1.32 ± 0.03 ^JKc	2.12 ± 0.07 ^Bb	0.35 ± 0.02 ^Mc	3.87 ± 0.47 ^Id	2.82 ± 0.25 ^Ke
	70% EtOH	8.6 ± 0.45 ^Le	3.15 ± 0.01 ^FGc	1.9 ± 0.05 ^Cb	0.4 ± 0.01 ^Md	6.63 ± 0.58 ^DEb	3.63 ± 0.3 ^Ic
	EA	7.26 ± 0.03 ^Ma	18.23 ± 0.17 ^Aa	-	8.22 ± 0.18 ^Aa	0.48 ± 0.04 ^KLb	-
Conventional Extraction
Cystophora sp.	70% ACE	18.27 ± 1.06 ^Ba	2.46 ± 0.03 ^Da	0.15 ± 0.01 ^FGc	0.57 ± 0.03 ^HIa	0.48 ± 0.21 ^Ke	7.17 ± 0.55 ^Db
	70% MeOH	18.59 ± 0.54 ^Aa	1.83 ± 0.01 ^Ea	2.55 ± 0.07 ^Ba	0.94 ± 0.26 ^Fb	7.35 ± 0.1 ^Aa	8.94 ± 0.42 ^Ba
	70% EtOH	15.49 ± 0.59 ^Ca	2.48 ± 0.04 ^Da	1.40 ± 0.07 ^Da	0.79 ± 0.14 ^Gb	6.2 ± 0.25 ^Cb	8.44 ± 0.55 ^Ca
	EA	0.96 ± 0.05 ^La	2.51 ± 0.15 ^Cc	0.17 ± 0.02 ^FGb	-	-	-
Sargassum sp.	70% ACE	13.93 ± 0.45 ^D	0.34 ± 0.01 ^Ke	0.28 ± 0.03 ^EFb	0.65 ± 0.01 ^Ha	4.74 ± 0.15 ^Ea	3.05 ± 0.13 ^Gd
	70% MeOH	10.37 ± 0.63 ^Fc	1.38 ± 0.02 ^Kb	0.43 ± 0.11 ^Ec	0.54 ± 0.03 ^HIc	7.03 ± 0.08 ^Bb	2.91 ± 0.19 ^Hb
	70% EtOH	8.89 ± 0.19 ^Gc	0.96 ± 0.06 ^Kd	0.13 ± 0.01 ^FGc	0.58 ± 0.01 ^HIc	3.7 ± 0.06 ^Fd	0.69 ± 0.2 ^Kd
	EA	0.53 ± 0.03 ^Mc	6.35 ± 0.01 ^Bb	0.02 ± 0.01 ^Gc	6.32 ± 0.8 ^Bb	0.12 ± 0.06 ^Lc	-
Phyllospora comosa	70% ACE	13.72 ± 0.50 ^Db	1.07 ± 0.02 ^Fb	0.59 ± 0.01 ^Hd	0.38 ± 0.01 ^JKb	2.26 ± 0.35 ^Id	1.87 ± 0.12 ^Je
	70% MeOH	11.03 ± 0.81 ^Eb	0.22 ± 0.01 ^Lc	0.14 ± 0.05 ^EFd	0.33 ± 0.01 ^Kd	1.48 ± 0.22 ^Jd	1.92 ± 0.01 ^Jd
	70% EtOH	10.28 ± 0.59 ^Fb	0.91 ± 0.02 ^Gb	-	0.47 ± 0.03 ^IJd	6.95 ± 0.09 ^Ba	4.69 ± 0.2 ^Eb
	EA	0.68 ± 0.01 ^Mc	6.52 ± 0.03 ^Aa	-	6.01 ± 0.01 ^Cc	2.22 ± 0.2 ^Ia	-
Ecklonia radiata	70% ACE	10.78 ± 0.31 ^Gc	0.35 ± 0.03 ^Jd	-	0.59 ± 0.03 ^HIa	2.6 ± 0.05 ^Hc	3.6 ± 0.3 ^Fc
	70% MeOH	10.63 ± 0.38 ^Hd	0.12 ± 0.04 ^Lc	-	1.12 ± 0.12 ^Ea	1.46 ± 0.1 ^Jd	2.5 ± 0.04 ^Ic
	70% EtOH	8.74 ± 0.21 ^Id	0.34 ± 0.05 ^Gb	-	0.91 ± 0.07 ^FGa	4.94 ± 0.7 ^Dc	2.82 ± 0.02 ^Hc
	EA	-	0.11 ± 0.02 ^Aa	27.54 ± 0.23 ^Aa	6.57 ± 0.3 ^Aa	0.46 ± 0.3 ^Kb	-
Durvillaea sp.	70% ACE	8.13 ± 0.20 ^Hd	0.99 ± 0.03 ^Ic	0.8 ± 0.22 ^Da	0.32 ± 0.07 ^Kb	2.8 ± 0.09 ^Gb	20.04 ± 0.23 ^Aa
	70% MeOH	4.32 ± 0.15 ^Ke	0.04 ± 0.01 ^Md	1.61 ± 0.09 ^Cb	0.29 ± 0.07 ^Kd	6.19 ± 0.01 ^Cc	0.19 ± 0.01 ^Le
	70% EtOH	5.01 ± 0.33 ^Je	0.78 ± 0.01 ^Hc	0.53 ± 0.07 ^Eb	0.39 ± 0.05 ^JKe	4.8 ± 0.07D ^Ec	2.91 ± 0.26 ^Hc
	EA	0.09 ± 0.01 ^Nd	6.35 ± 0.01 ^Bc	-	0.9 ± 0.01 ^FGe	-	-

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All values are expressed as the mean ± SD and performed in triplicates. Different letters (^a,b,c,d,e) within the same column are significantly different (p < 0.05) samples within the solvent whereas letters (^A–Q) within the same column are significantly different (p < 0.05) samples within the species. Six species of seaweed are reported based on dry weight. CE (catechin equivalents), QE (quercetin equivalents), GAE (gallic acid equivalents), PGE (phloroglucinol equivalents). TFC (total flavonoids content), TPC (total phenolic content), TCT (total tannins content), DMBA (2,4-dimethoxybenzaldehyde assay), PBA (Prussian blue assay), FDA (Folin–Denis Assay). The abbreviation of solvents expressed are ACE (70% Acetone), MeOH (70% Methanol), EtOH (70% Ethanol), EA (Absolute Ethyl Acetate).

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.

In our study, the total tannin content ranged from 0.27 mg CE/g (Sargassum sp., 70% acetone) to 2.86 mg CE/g (Phyllospora comosa, 70% methanol) in ultrasonication method whereas in conventional method the total tannin content ranged from 0.02 mg CE/g (Phyllospora comosa, 70% acetone) to 27.54 mg CE/g (Ecklonia radiata, absolute ethyl acetate). Tannin content was not observed in solvents other than ethyl acetate during the conventional extraction of Ecklonia radiata. Tannin content was observed only in 70% methanol and 70% ethanol solvents in the ultrasonication method. However, surprisingly, the highest tannin content among all samples and methods was obtained from conventional extraction with absolute ethyl acetate of Ecklonia radiata. The tannin content from the brown seaweed Ecklonia radiata (27.54 mg CE/g, absolute ethyl acetate) was significantly higher than (p < 0.05) Cystophora sp. (2.55 mg CE/g, 70% methanol), Durvillaea sp. (1.61 mg CE/g, 70% methanol), Sargassum sp. (0.43 mg CE/g, 70% methanol) and Phyllospora cosmosa (0.34 mg CE/g, 70% methanol) when extracted by conventional method, whereas the brown seaweed, Phyllospora comosa (2.86 mg, 70 % methanol) was significantly higher than (p < 0.05) Durvillaea sp. (2.12 mg CE/g, 70% methanol), Ecklonia radiata (1.29 mg CE/g, 70% acetone), Cystophora sp. (0.94 mg CE/g, 70% ethanol), Sargassum sp. (0.27 mg CE/g, 70% acetone) in ultrasonication methodology. The difference in tannin content among different solvents is due to the solubility of tannin in the solvent, as well as the molecular weight and degree of polymerization [29]. In a previous study, conventionally extracted Sargassum sp. and Ecklonia sp. using 80% ethanol were estimated to have 5.62 µg CE/g and 166.87 µg CE/g, respectively [20]. The difference in tannin content between the two seaweeds may be due to variations in light intensity exposure, size, ultraviolet radiation, species, age, and salinity [25].

The phlorotannin content were estimated using three assays, DMBA, FDA, and PBA. In the DMBA assay, the absolute ethyl acetate extracted ultrasonication methodology of Ecklonia radiata (8.29 PGE mg/g) and Durvillaea sp. (8.22 PGE mg/g) were significantly higher compared to other solvents and species. The highest concentration of phlorotannin was extracted using conventional methodology from absolute ethyl acetate Ecklonia radiata (6.57 PGE mg/g), followed by Sargassum sp. (6.32 PGE mg/g) and Phyllospora comosa (6.01 PGE mg/g). In PBA, ultrasonication extraction of Phyllospora comosa (12.62 PGE mg/g, 70% methanol) yielded higher phlorotannin content than Cytosphora sp. (8.61 PGE mg/g, 70% methanol) and Cytosphora sp. (8.11 PGE mg/g, 80% ethanol). In comparison, conventional extraction resulted in the highest phlorotannin content for Cytosphora sp. (7.35 PGE mg/g, 70% methanol), followed by Sargassum sp. (7.03 PGE mg/g, 70% methanol), Phyllospora comosa (6.95 PGE mg/g, 70% ethanol), Cytosphora sp. (6.2 PGE mg/g, 70% ethanol), and Durvillaea sp. (6.19 PGE mg/g, 70% methanol). In the FDA, 70% ethanol extracted Cystophora sp. (14.2 PGE mg/g) exhibited the highest presence of phlorotannin in ultrasonication, followed by Phyllospora comosa (8.57 PGE mg/g, 70% acetone) and Sargassum sp. (7.64 PGE mg/g, 70% acetone). However, conventional extraction of Durvillaea sp. with 70% acetone resulted in a higher phlorotannin content (20.04 PGE mg/g) than ultrasonication. Overall, ultrasonication extracted more phlorotannin compounds, and the solvents used to extract phlorotannin content varied among the phlorotannin assays. The reason for this variation might be due to the slightly different mechanisms of the assays, which interact with the target compounds differently and have varying sensitivities [30].

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.

2.2. 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].

Table 2.

Estimation of freeze-dried antioxidant potential of seaweed species extracted by the conventional and non-conventional method of ultrasonication.

Samples	Solvents	DPPH (mg TE/g)	FRAP (mg TE/g)	ABTS (mg TE/g)	FICA (mg EDTA/g)	·OH-RSA (mg TE/g)	TAC (mg TE/g)	RPA (mg TE/g)
Ultrasonication
Cystophora sp.	70% ACE	50.33 ± 0.07 ^Aa	45.62 ± 0.13 ^Aa	64.15 ± 0.20 ^Aa	1.76 ± 0.01 ^BCd	13.26 ± 0.22 ^Gd	76.61 ± 0.30 ^Aa	17.21 ± 0.03 ^Gc
	70% MeOH	43.6 ± 0.10 ^Ba	26.08 ± 0.22 ^Ba	58.65 ± 0.18 ^Ba	0.96 ± 0.01 ^Fd	62.67 ± 0.44 ^Bb	29.23 ± 1.04 ^Ic	16.11 ± 0.08 ^Hc
	70% EtOH	7.26 ± 0.03 ^Hb	25.00 ± 0.22 ^Ca	44.24 ± 0.18 ^Db	1.04 ± 0.01 ^Fa	-	33.30 ± 1.20 ^Gb	29.27 ± 0.14 ^Aa
	EA	1.42 ± 0.8 ^Nb	0.35 ± 0.01 ^Nc	6.58 ± 0.06 ^Pb	1.45 ± 0.9 ^BCb	9.06 ± 0.55 ^I	20.24 ± 0.6 ^Kc	1.95± 0.35 ^Pb
Phyllospora comosa	70% ACE	37.52 ± 0.07 ^Dc	17.01 ± 0.12 ^Gc	22.99 ± 0.85 ^Kb	4.14 ± 0.11 ^Aa	53.38 ± 1.10 ^Cb	48.71 ± 0.76 ^Cc	25.76 ± 0.09 ^Cb
	70% MeOH	4.76 ± 0.03 ^Le	7.80 ± 0.23 ^Ic	33.22 ± 0.33 ^Fc	1.91 ± 0.02 ^Ba	65.42 ± 0.96 ^Ba	33.52 ± 0.60 ^Ga	20.46 ± 0.18 ^Da
	70% EtOH	4.69 ± 0.01 ^Kd	11.02 ± 0.19 ^Hd	32.21 ± 0.11 ^Ge	1.59 ± 0.01 ^B–Db	15.38 ± 0.17 ^Fa	40.94 ± 0.92 ^Da	7.24 ± 0.04 ^Me
	EA	2.1 ± 0.03 ^Ma	0.53 ± 0.04 ^Nd	9.03 ± 0.03 ^Oa	1.02 ± 0.04 ^Fb	33.23 ± 0.15 ^H	-	2.90 ± 0.04 ^Oa
Sargassum sp.	70% ACE	38.79 ± 0.06 ^Cb	36.73 ± 0.27 ^Db	45.16 ± 0.40 ^Cd	1.45 ± 0.01 ^C–Ed	16.73 ± 0.36 ^Ec	56.01 ± 0.35 ^Bb	28.47 ± 0.14 ^Ba
	70% MeOH	7.89 ± 0.06 ^Ga	35.60 ± 0.21 ^Eb	30.13 ± 0.07 ^Hd	1.11 ± 0.01 ^Fc	3.66 ± 0.01 ^Fd	32.58 ± 0.63 ^GHb	15.53 ± 0.04 ^Id
	70% EtOH	7.84 ± 0.03 ^Ga	27.63 ± 0.16 ^Fb	30.22 ± 0.75 ^Ha	1.01 ± 0.01 ^Ga	1.33 ± 0.05 ^Kd	31.63 ± 0.46 ^Hc	18.52 ± 0.19 ^Fb
	EA	0.62 ± 0.01 ^Oc	0.09 ± 0.01 ^Mb	4.86 ± 0.05 ^Qd	0.52 ± 0.02 ^Hc	-	40.30 ± 1.26 ^Ea	-
Ecklonia radiata	70% ACE	34.09 ± 0.05 ^Ed	24.10 ± 0.12 ^Db	24.92 ± 0.45 ^Jd	1.87 ± 0.01 ^Bb	134.49 ± 5.25 ^Aa	37.82 ± 1.5 ^Fd	11.90 ± 0.04 ^Jd
	70% MeOH	5.01 ± 0.01 ^Ld	20.80 ± 0.09 ^Eb	40.91 ± 0.99 ^Eb	0.53 ± 0.01 ^He	11.94 ± 0.01 ^Cb	15.35 ± 0.76 ^Ld	19.10 ± 0.08 ^Eb
	70% EtOH	5.51 ± 0.02 ^Ic	19.20 ± 0.16 ^Fb	21.09 ± 0.69 ^Lc	1.87 ± 0.01 ^D–Fa	3.23 ± 0.06 ^Jc	26.43 ± 1.5 ^Jd	8.61 ± 0.08 ^Ld
	EA	-	0.84 ± 0.01 ^Mb	6.75 ± 0.10 ^PQc	0.66 ± 0.01 ^Ba	-	33.79 ± 0.42 ^Hb	1.88 ± 0.02 ^Qc
Durvillaea sp.	70% ACE	22.49 ± 0.04 ^Fe	3.58 ± 0.08 ^Ld	28.42 ± 0.07 ^Ie	1.06 ± 0.01 ^Fe	13.12 ± 0.16 ^Gd	18.50 ± 0.08 ^Ke	4.68 ± 0.16 ^Ne
	70% MeOH	5.24 ± 0.01 ^Jc	6.39 ± 0.12 ^Jd	17.67 ± 0.09 ^Ne	1.16 ± 0.01 ^EFb	18.22 ± 0.02 ^Dc	7.01 ± 0.15 ^Ne	11.99 ± 0.04 ^Je
	70% EtOH	5.47 ± 0.01 ^Ic	11.39 ± 0.08 ^Hc	18.36 ± 0.40 ^Mc	1.01 ± 0.01 ^Fa	14.27 ± 0.09 ^Fb	9.36 ± 0.15 ^Me	11.65 ± 0.06 ^Kc
	EA	2.01 ± 0.01 ^Ma	0.25 ± 0.01 ^Ka	6.40 ± 0.03 ^Qd	1.92 ± 0.01 ^Ba	-	5.86 ± 0.05 ^Od	2.00 ± 0.03 ^Pb
Conventional Extraction
Cystophora sp.	70% ACE	46.73 ± 0.12 ^Aa	32.54 ± 0.29 ^Ca	42.50 ± 0.22 ^Aa	0.24 ± 0.01	0.70 ± 0.73 ^Jc	77.63 ± 0.17 ^Aa	7.92 ± 0.03 ^Fd
	70% MeOH	42.95 ± 0.12 ^Ca	32.88 ± 0.12 ^Ba	41.10 ± 0.15 ^Ba	0.10 ± 0.01 ^Nc	2.20 ± 0.74 ^Hc	46.00 ± 0.54 ^Ca	15.20 ± 0.14 ^Ba
	70% EtOH	46.49 ± 0.08 ^Ba	33.48 ± 0.23 ^Aa	42.34 ± 0.32 ^Aa	0.37 ± 0.01 ^Ke	3.72 ± 0.05 ^Gc	73.79 ± 0.30 ^Ba	22.01 ± 0.10 ^Aa
	EA	4.40 ± 0.06 ^Jb	0.25 ± 0.01 ^Opb	3.41 ± 0.04 ^Lb	0.93 ± 0.01 ^Ee	-	17.59 ± 0.35 ^Ha	2.27 ± 0.09 ^La
Sargassum sp.	70% ACE	32.94 ± 0.17 ^Ec	30.35 ± 0.17 ^Db	23.43 ± 0.27 ^Cb	0.36 ± 0.02 ^Kc	23.37 ± 0.74 ^Ca	34.75 ± 0.52 ^Cb	10.55 ± 0.08 ^Da
	70% MeOH	5.97 ± 0.08 ^Hc	17.83 ± 0.23 ^Fb	23.34 ± 0.48 ^Cb	0.46 ± 0.01 ^Jc	3.60 ± 0.29 ^Gd	8.85 ± 0.17 ^Dd	7.84 ± 0.05 ^Hd
	70% EtOH	5.87 ± 0.07 ^Hc	16.96 ± 0.32 ^Gb	21.53 ± 0.48 ^Eb	0.48 ± 0.03 ^Jc	0.39 ± 0.35 ^Jd	14.77 ± 0.18 ^Ld	8.91 ± 0.08 ^Fc
	EA	3.44 ± 0.19 ^Kc	0.40 ± 0.01 ^Oa	2.88 ± 0.03 ^Mc	0.35 ± 0.02 ^Kd	-	16.64 ± 1.30 ^Ib	0.43 ± 0.01 ^Mb
Phyllospora comosa	70% ACE	35.09 ± 0.09 ^Db	12.69 ± 0.23 ^Hd	20.24 ± 0.87 ^Fd	0.98 ± 0.01 ^Da	5.80 ± 0.01 ^Eb	18.01 ± 0.63 ^Jd	8.27 ± 0.04 ^Gc
	70% MeOH	4.99 ± 0.01 ^Id	6.56 ± 0.12 ^Ld	12.56 ± 0.10 ^Ijd	0.78 ± 0.01 ^Fa	26.63 ± 0.12 ^Aa	13.71 ± 0.31 ^Ic	9.94 ± 0.05 ^Eb
	70% EtOH	5.12 ± 0.01 ^Hd	9.18 ± 0.29 ^Kd	12.66 ± 0.07 ^Ijd	1.19 ± 0.01 ^Ba	4.51 ± 0.13 ^Fb	21.69 ± 0.09 ^Fa	11.90 ± 0.12 ^Cb
	EA	0.69 ± 0.01 ^Me	0.17 ± 0.01 ^Opb	2.85 ± 0.05 ^Ka	1.02 ± 0.01 ^Cb	-	-	0.44 ± 0.02 ^Mb
Ecklonia radiata	70% ACE	41.96 ± 0.07 ^Fd	20.88 ± 0.12 ^Ec	25.35 ± 0.62 ^Dc	0.66 ± 0.01 ^Ib	2.17 ± 0.05 ^H	27.91 ± 0.17 ^Fc	8.71 ± 0.07 ^Ge
	70% MeOH	31.59 ± 0.08 ^Fb	12.32 ± 0.12 ^Ic	19.62 ± 0.40 ^Hc	0.37± 0.01 ^Ld	6.75 ± 0.15 ^Dc	28.09 ± 0.15 ^Eb	10.17 ± 0.07 ^Fc
	70% EtOH	29.41 ± 0.12 ^Gb	14.06 ± 0.19 ^Hc	13.75 ± 0.23 ^Ijc	0.66 ± 0.01 ^Jd	1.11 ± 0.65 ^Ie	28.24 ± 0.23 ^Gc	11.43 ± 0.04 ^Fc
	EA	2.51 ± 0.04 ^Ld	0.02 ± 0.01 ^Pc	-	4.56 ± 0.07 ^Aa	-	-	0.14 ± 0.01 ^Nd
Durvillaea sp.	70% ACE	30.54 ± 0.06 ^Fe	10.25 ± 0.08 ^Je	12.86 ± 0.07 ^Ie	0.27 ± 0.01 ^Md	4.56 ± 0.14 ^Ec	13.31 ± 0.09 ^Ke	9.84 ± 0.04 ^Ec
	70% MeOH	4.73 ± 0.02 ^Je	6.20 ± 0.20 ^Me	12.60 ± 0.07 ^Ijd	0.56 ± 0.02 ^Hb	16.80 ± 0.15 ^Bb	4.09 ± 0.04 ^Ne	5.12 ± 0.04 ^Je
	70% EtOH	4.77 ± 0.02 ^Je	5.83 ± 0.16 ^Ne	12.29 ± 0.04 ^Je	0.66 ± 0.01 ^Gb	6.50 ± 0.23 ^Da	5.34 ± 0.14 ^Me	4.51 ± 0.03 ^Kd
	EA	4.82 ± 0.03 ^Ja	0.03 ± 0.01 ^Pc	1.87 ± 0.04 ^Ne	0.93 ± 0.01 ^Ec	-	8.01 ± 0.53 ^Lc	-

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All values are expressed as the mean ± SD and performed in triplicates. Different letters (^a,b,c,d,e,j) within the same column are significantly different (p < 0.05) samples within the solvent whereas letters (^A–Q) within the same column are significantly different (p < 0.05) samples within the species. Six species of seaweed are reported based on dry weight. TE (Trolox equivalents), EDTA (ethylenediaminetetraacetic acid), FRAP (ferric reducing antioxidant power), DPPH (2,2′-diphenyl-1-picrylhydrazyl), TAC (total antioxidant capacity), ABTS (2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid), RPA (reducing power assay), ·OH-RSA (hydroxyl radical scavenging activity), FICA (ferrous ion chelating activity). The abbreviation of solvents expressed are ACE (Acetone), MeOH (Methanol), EtOH (Ethanol), EA (Ethyl Acetate).

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.

In RPA, the ultrasound extraction method revealed that the order of antioxidant potential were Cystophora sp. (29.27 mg TE/g, 70% ethanol), followed by Sargassum sp. (28.47 mg TE/g, 70% acetone), Phyllospora comosa (25.76 mg TE/g, 70% acetone), Phyllospora comosa (20.46 mg TE/g, 70% methanol), Ecklonia radiata (19.1 mg TE/g, 70% methanol) and Sargassum sp. (18.52 mg TE/g, 70% ethanol). On the other hand, the conventional method yielded lower antioxidant activity, with Cystophora sp. (22.01 mg TE/g, 70% ethanol) having the highest potency, followed by Phyllospora comosa (11.90 mg TE/g, 70% ethanol), Ecklonia radiata (11.43 mg TE/g, 70% ethanol), Sargassum sp. (10.55 mg TE/g, 70% acetone), Ecklonia radiata (10.17 mg TE/g, 70% methanol), and Durvillaea sp. (9.84 mg TE/g, 70% acetone). Therefore, it can be concluded that the ultrasound method is more effective for extracting antioxidants, with 70% acetone, 70% methanol, and 70% ethanol being the recommended solvents for extraction.

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.

2.3. 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.

Variables	TPC	TFC	TCT	DMBA	PBA	FDA	DPPH	FRAP	ABTS	FICA	OH-RSA	TAC
Ultrasonication
TFC	−0.178
TCT	0.222	−0.155
DMBA	−0.469	0.491 ^b	−0.414
PBA	0.649 ^a	−0.238	0.394	−0.663
FDA	0.818 ^a	−0.297	0.422	−0.421	0.432
DPPH	0.505 ^b	−0.349	−0.170	−0.236	0.178	0.498 ^b
FRAP	0.731 ^a	−0.365	−0.145	−0.503	0.403	0.600 ^a	0.657 ^a
ABTS	0.837 ^a	−0.245	0.110	−0.571	0.510 ^b	0.734 ^a	0.690 ^a	0.815 ^a
FICA	0.202	−0.067	0.118	0.150	0.114	0.279	0.406	0.111	0.016
OH-RSA	0.165	−0.270	0.254	−0.164	0.311	0.024	0.474 ^b	0.114	0.161	0.375
TAC	0.448 ^b	−0.442	−0.111	−0.193	0.110	0.571 ^a	0.646 ^a	0.650 ^a	0.562 ^a	0.326	0.120
RPA	0.819 ^a	−0.294	0.286	−0.458	0.682 ^a	0.771 ^a	0.480 ^b	0.712 ^a	0.699 ^a	0.313	0.182	0.449 ^b
Conventional
TFC	−0.434
TCT	−0.292	−0.205
DMBA	−0.668	0.511 ^b	0.488
PBA	0.457 ^b	−0.384	−0.196	−0.513
FDA	0.478 ^b	−0.195	−0.133	−0.393	0.262
DPPH	0.799 ^a	−0.262	−0.186	−0.445	0.167	0.561 ^b
FRAP	0.907 ^a	−0.304	−0.217	−0.538	0.478 ^a	0.447 ^b	0.824 ^a
ABTS	0.921 ^b	−0.280	−0.257	−0.567	0.457 ^a	0.451 ^b	0.809 ^a	0.958 ^a
FICA	−0.460	−0.122	0.926 ^a	0.537 ^b	−0.298	−0.321	−0.376	−0.443	−0.480
OH-RSA	0.247	−0.444	−0.153	−0.390	0.190	−0.40	−0.021	0.112	0.047	−0.168
TAC	0.761 ^a	0.055 ^b	−0.220	−0.317	0.154	0.403	0.809 ^a	0.837 ^a	0.851 ^a	−0.393	−0.063
RPA	0.830 ^a	−0.449	−0.273	−0.625	0.628 ^a	0.541 ^b	0.688 ^a	0.785 ^a	0.787 ^a	−0.423	0.219	0.685 ^a

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^a Significant correlation with p ≤ 0.01; ^b Significant correlation with p ≤ 0.05.

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).

Principal component analysis (PCA) of the phenolic content (TPC, TFC, TCT, DMBA, PBA, FDA) and antioxidant activities (DPPH, ABTS, FRAP, RPA, OH-RSA, TAC) of five brown seaweeds extracted via ultrasonication (A) and conventional (B) methodologies.

2.4. Distribution of Phenolic Compounds in Seaweeds

Seaweed contains an extensive range of phenolic compounds in different conjugated forms. A Venn diagram can make it easier to see the distribution of the phenolic compounds. The Venn diagrams (Figure 2) were developed according to the number of phenolic compounds that were detected among the five species of seaweed including Phyllospora comosa (blue), Ecklonia radiata (red), Durvillaea sp. (green), Sargassum sp. (yellow), and Cystophora sp. (brown). In the Venn diagram (Figure 2A), the unique compounds present in Cystophora sp., Durvillaea sp., Phyllospora comosa, Ecklonia radiata, Sargassum sp. were 7, 7, 18, 21, and 64, respectively. The maximum overlapping phenolics were 122 among the 5 species of seaweed. The lowest number of phenolics that overlapped were Cystophora sp., and Ecklonia radiata. A study reported that the seaweed species collected from the shores of Australia and New Zealand, including Phyllospora sp., Ecklonia radiata, Durvillaea sp., Sargassum sp., Cystophora sp., had high total phenolic content [43]. To support our study, research identified a wide range of phenolic compounds in Cystophora sp. and Sargassum sp. [44]. In Figure 2B, 26 phenolic acids were common among the 5 seaweed species. The unique compounds in Ecklonia radiata, Cystophora sp., Durvillaea sp., Phyllospora comosa and Sargassum sp., were 2, 3, 4, 7, and 24, respectively. In Venn diagram (Figure 2C), 33 compounds were common flavonoids among the 5 species of seaweed. The unique flavonoids were 1, 2, 3, 4, and 34 in Durvillaea sp., Phyllospora comosa, Ecklonia radiata, Cystophora sp., and Sargassum sp., respectively. The highest flavonoids of 26 compounds overlapped with Phyllospora comosa, Durvillaea sp., Sargassum sp., and Cystophora sp. The unique other phenolics (Figure 2D) in Phyllospora comosa, Sargassum sp., Cystophora sp., Durvillaea sp., and Ecklonia radiata were 1, 2, 6, 8, and 17, respectively. The 41 compounds of all 5 seaweed species were overlapped. The highest overlapped compounds were 29, in Durvillaea sp., Ecklonia radiata, Sargassum sp., and Cystophora sp.

Venn diagram of phenolic compounds presented in different seaweed species. (A) shows the relation of total polyphenols among the species (B) shows the relation of phenolic acids among the species. (C) shows the relation of flavonoids among the species. (D) shows the relation between the other polyphenols in different species.

The Venn diagrams (Figure 3) were developed to illustrate the number of phenolic compounds extracted using different solvents, including 70% ethanol (yellow), absolute ethyl acetate (green), 70% methanol (red), and 70% acetone (blue). In the Venn diagram of total phenolics (Figure 3A) extracted using different solvents were 2 (0.4%), 4 (0.9%), 13 (2.8%), and 85 (18.2%), of the unique compounds in absolute ethyl acetate, 70% methanol, 70% acetone, and 70% ethanol, respectively. A study supported our results showed ethanol extracted a higher amount of phenolic compounds, when compared with methanol or acetone [45]. The reason might be because ethanol has higher polarity and so improved phenolic solubility [46]. In another study, 80% methanol and 80% ethanol exhibited higher phenolic content [47]. The overlapped compounds among the 4 solvents were 189 (40.6%) compounds. A total of 102 (21.9%) compounds were common among 70% methanol, 70% ethanol, and 70% acetone. The lowest compounds were overlapped with 70% ethanol and absolute ethyl acetate. The phenolic acids that were common between 4 solvents were 37 (26.8%) compounds (Figure 3B). The unique compounds were only high in 70% ethanol solvent consisting of 54 (39.1%) compounds. A total of 18 (13%) compounds were shared between 70% ethanol, 70% methanol, and 70% acetone. A total of 12 (8.7%) compounds were shared between 70% acetone and 70% methanol. The overlapped flavonoids (Figure 3C) among the solvent were 76 (32.2%) compounds. The unique compounds were 3 (1.3%), 9 (3.8%), 19 (8.1%) and 38 (16.1%), in 70% methanol, 70% acetone, absolute ethyl acetate and 70% ethanol, respectively. Flavonoids of 64 compounds (27.1%) were common between 70% ethanol, 70% methanol, and 70% acetone. Another study reported that the ethanol exhibited high flavonoid content and extracted lower levels with acetone, which is consistent with the results of our study [48]. In other polyphenols (Figure 3D), the unique compounds were 7 (4.6%), 9 (5.9%) and 23 (15.1%), in 70% ethanol, absolute ethyl acetate, and 70% acetone, respectively. The overlapped compounds were 60 (39.5%) among the 4 solvents. A total of 27 (17.8%) compounds were shared among the 70% ethanol, 70% methanol, and 70% acetone.

Venn diagram of phenolic compounds presented in different extracted solvents. (A) shows the relation of total phenolic compounds present in different solvents used in extraction. (B) shows the relations of phenolic acids among the solvents (C) shows the relations of flavonoids present in different solvents. (D) shows the other polyphenols present in solvents.

The Venn diagrams (Figure 4) were developed according to the number of phenolic compounds extracted via the methodologies of conventional (blue) and ultrasonication (yellow). In the Venn diagram (Figure 4A), the overlapped compounds of conventional and ultrasonication were 332 (71.2%). The unique phenolic compounds were 20 (4.3%) and 114 (24.5%) in conventional and ultrasonication. The phenolic acids (Figure 4B) common to both methodologies were 70 (65.4%) compounds. The phenolic acids unique to conventional and ultrasonication were 5 (4.7%) and 32 (29.9%), respectively. The overlapped flavonoids (Figure 4C) were 161 (66%), unique flavonoids were 11 (4.5%), and 72 (29.5%) in conventional and ultrasonication extraction, respectively. A previous study observed flavonoids were high after ultrasonication extraction, which is consistent with our results [49]. The other polyphenols (Figure 4D) in conventional and ultrasonication were 8 (5.3%) and 40 (26.3%), respectively. However, the unique compounds of total polyphenols were 104 (68.4%) compounds. Ultrasonication methodology extracts low molecular weight compounds by disrupting cell walls and transfer mass of the phenolics. This might be one of the reasons that our study also observed high phenolic compound extracted by ultrasonication [50].

Venn diagram of phenolic compounds extracted via the methodologies of ultrasonication and conventional. (A) shows the relations of total polyphenols in different methodologies. (B) shows relations of phenolic acids in different methodologies. (C) shows relations of flavonoids in different methodologies. (D) shows relations of other polyphenols in different methodologies.

In Figure 5A, the Venn diagram of the bound and free total phenolics of the overlapped phenolics were 320 (68.8%), whereas the bound and free unique total phenolics were 34 (7.3%) and 111 (23.9%), respectively. The overlapped phenolic acids (Figure 5B) were 65 (60.7%). However, the bound and free phenolic acids were 10 (9.3%) and 32 (29.9%) compounds, respectively. The overlapped flavonoids (Figure 5C) were 155 (63.8%) whereas the unique bound and free were 39 (16%) and 49 (20.2%) compounds, respectively. The other polyphenols (Figure 5D) in bound and free unique compounds were 19 (12.5%) and 32 (21.1%), respectively. However, the overlapped compounds were 101 (66.4%). Harukaze et al. [51] reported higher bound phenolics than the free phenolics.

Venn diagram presented in free and bound extraction. (A) shows the relations of total phenolic compounds present in free and bound phenolics (B) shows the relation of phenolic acids in free and bound phenolics (C) shows the relation of flavonoids in free and bound phenolics. (D) shows the relation of other polyphenols in free and bound phenolics.

2.5. 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 (Table 4 and Table 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 MS² in ultrasonication and conventional, respectively.

Table 4.

Characterization of phenolic compounds in ultrasound extracted seaweed samples by LC-ESI-QTOF-MS/MS.

No.	Proposed Compounds	Molecular Formula	RT (min)	Ionization (ESI⁺/ESI⁻)	Molecular Weight	Theoretical (m/z)	Observed (m/z)	Error (ppm)	MS² Product Ions	Seaweed Samples
Phenolic acid
Hydroxybenzoic acids
1	4-Hydroxybenzaldehyde	C₇H₆O₂	7.188	[M – H]⁻	122.0376	121.0303	121.0303	0.2	77	Dabu
2	Protocatechuic acid 4-O-glucoside	C₁₃H₁₆O₉	14.994	[M – H]⁻	316.0786	315.0713	315.0702	−3.5	153	* Embu, Dmbu, Eeu
3	2,3-Dihydroxybenzoic acid	C₇H₆O₄	25.802	[M – H]⁻	154.0262	153.0189	153.0190	0.7	109	Eethbu
4	Gallic acid 4-O-glucoside	C₁₃H₁₆O₁₀	30.462	** [M – H]⁻	332.0768	331.0695	331.0691	−1.2	169, 125	* Amu, Dmu, Aeu, Aau, Cabu, Cebu, Dmbu
5	Gallic acid	C₇H₆O₅	31.309	[M – H]⁻	170.0225	169.0152	169.0154	1.2	125	* Aabu, Aebu, Ambu, Babu, Bebu, Bethbu, Bmbu, Dethbu, Eabu, Eethbu, Eeu, Eau
6	2-Hydroxybenzoic acid	C₇H₆O₃	32.019	[M – H]⁻	138.0310	137.0237	137.0238	0.7	93	* Dabu, Dmbu
Hydroxycinnamic acids
7	Feruloyl tartaric acid	C₁₄H₁₄O₉	4.933	[M – H]⁻	326.0652	325.0579	325.0575	−1.2	193, 149	* Eau, Cau
8	m-Coumaric acid	C₉H₈O₃	5.228	[M – H]⁻	164.0487	163.0414	163.0412	−1.2	119	* Dabu, Cmbu, Debu, Dmbu, Eabu
9	Caffeoyl tartaric acid	C₁₃H₁₂O₉	5.426	[M – H]⁻	312.0504	311.0431	311.0438	2.3	161	* Emu, Dmu, Amu
10	Ferulic acid	C₁₀H₁₀O₄	5.539	[M – H]⁻	194.0585	193.0512	193.0516	2.1	178, 149, 134	Eau
11	Isoferulic acid 3-sulfate	C₁₀H₁₀O₇S	5.608	[M – H]⁻	274.0129	273.0056	273.0054	−0.7	193, 178	* Aabu, Aethbu, Ambu, Bmbu, Cabu, Dabu, Amu
12	Caffeic acid 3-O-glucuronide	C₁₅H₁₆O₁₀	6.388	** [M – H]⁻	356.0743	355.0670	355.0673	0.8	179	* Cebu, Ambu, Embu, Cethbu, Emu, Amu
13	Hydroxycaffeic acid	C₉H₈O₅	7.205	[M – H]⁻	196.0368	195.0295	195.0299	2.1	151	Cebu
14	Cinnamic acid	C₉H₈O₂	7.246	[M – H]⁻	148.0537	147.0464	147.0465	0.7	103	* Cebu, Aethbu, Bmbu, Cabu, Cmbu, Dabu, Eabu
15	Ferulic acid 4-O-glucoside	C₁₆H₂₀O₉	13.750	[M – H]⁻	356.1106	355.1033	355.1025	−2.3	193, 178, 149, 134	* Aabu, Babu
16	Chlorogenic acid	C₁₆H₁₈O₉	13.985	[M – H]⁻	354.0929	353.0856	353.0855	−0.3	253, 190, 144	* Dabu, Babu, Cabu, Cebu, Eabu, Eau, Aeu
17	1-Sinapoyl−2-feruloylgentiobiose	C₃₃H₄₀O₁₈	14.898	** [M – H]⁻	724.2205	723.2132	723.2136	0.6	529, 499	* Bmbu, Cabu, Cethbu, Cmbu, Dmbu, Eebu, Aeu
18	Sinapic acid	C₁₁H₁₂O₅	16.158	** [M – H]⁻	224.0666	223.0593	223.0595	0.9	205, 163	* Aabu, Cethbu, Dabu, Debu, Dmbu
19	p-Coumaroyl tartaric acid	C₁₃H₁₂O₈	16.214	[M – H]⁻	296.0555	295.0482	295.0486	1.4	115	* Debu, Dmbu, Embu
20	p-Coumaroyl malic acid	C₁₃H₁₂O₇	16.596	** [M – H]⁻	280.0596	279.0523	279.0512	−3.9	163, 119	* Dmbu, Cabu, Cebu, Cethbu, Dabu, Dmbu, Emu, Aeu
21	1,5-Dicaffeoylquinic acid	C₂₅H₂₄O₁₂	17.017	[M – H]⁻	516.1233	515.1160	515.1172	2.3	353, 335, 191, 179	* Dabu, Eabu, Eau
22	Ferulic acid 4-O-glucuronide	C₁₆H₁₈O₁₀	17.199	** [M – H]⁻	370.0895	369.0822	369.0836	3.8	193	* Eau, Bmu, Dabu, Debu, Embu
23	p-Coumaric acid 4-O-glucoside	C₁₅H₁₈O₈	17.367	[M – H]⁻	326.0970	325.0897	325.0891	−1.8	163	* Eabu, Dabu
24	Rosmarinic acid	C₁₈H₁₆O₈	18.249	[M – H]⁻	360.0850	359.0777	359.0775	−0.6	179	* Dmbu, Emu
25	Caffeoyl glucose	C₁₅H₁₈O₉	18.549	[M – H]⁻	342.0951	341.0878	341.0890	3.5	179, 161	* Dmbu, Dabu, Eabu
26	3-p-Coumaroylquinic acid	C₁₆H₁₈O₈	19.117	[M – H]⁻	338.0998	337.0925	337.0926	0.3	265, 173, 162	* Dabu, Cabu, Eabu, Eebu, Eau
27	p-Coumaric acid 4-O-glucoside	C₁₅H₁₈O₇	21.240	[M – H]⁻	310.1024	309.0951	309.0951	0.1	163	Debu
28	5-5′-Dehydrodiferulic acid	C₂₀H₁₈O₈	21.657	** [M – H]⁺	386.0985	385.0912	385.0913	0.3	369	* Eabu, Embu, Cebu
29	3-Sinapoylquinic acid	C₁₈H₂₂O₁₀	23.306	[M – H]⁻	398.1177	397.1104	397.1109	1.3	233, 179	Eeu
30	1,2,2′-Triferuloylgentiobiose	C₄₂H₄₆O₂₀	31.165	[M – H]⁻	870.2584	869.2511	869.2481	−3.5	693, 517	* Deu, Bau
Hydroxyphenyl acetic acids
31	3,4-Dihydroxyphenylacetic acid	C₈H₈O₄	6.661	[M – H]⁻	168.0432	167.0359	167.0350	−5.4	149, 123	* Deu
32	2-Hydroxy-2-phenylacetic acid	C₈H₈O₃	7.182	[M – H]⁻	152.0475	151.0402	151.0403	0.7	136, 92	* Cebu, Aeu, Aau
Hydroxyphenylpropanoic acids
33	Dihydroferulic acid 4-O-glucuronide	C₁₆H₂₀O₁₀	3.119	** [M – H]⁻	372.1090	371.1017	371.1019	0.5	195	* Babu, Bebu, Bmbu, Dabu, Eabu, Embu, Cethbu
34	Dihydrocaffeic acid 3-O-glucuronide	C₁₅H₁₈O₁₀	7.535	[M – H]⁻	358.0913	357.0840	357.0837	−0.8	181	* Bebu, Bmbu, Debu, Eabu, Eebu, Embu, Aabu
35	Dihydroferulic acid 4-sulfate	C₁₀H₁₂O₇S	16.763	[M – H]⁻	276.0290	275.0217	275.0212	−1.8	195, 151, 177	Eebu
Flavonoids
Flavanols
36	Theaflavin	C₂₉H₂₄O₁₂	3.082	[M – H]⁻	564.1254	563.1181	563.1198	3.0	545	* Bebu, Dabu, Eeu
37	(+)-Gallocatechin 3-O-gallate	C₂₂H₁₈O₁₁	5.144	[M – H]⁻	458.0818	457.0745	457.0744	−0.2	305, 169	* Cebu, Debu
38	Procyanidin dimer B1	C₃₀H₂₆O₁₂	17.032	[M – H]⁻	578.1385	577.1312	577.1292	−3.5	451	Babu
39	3′-O-Methylcatechin	C₁₆H₁₆O₆	17.951	** [M – H]⁻	304.0959	303.0886	303.0894	2.6	271, 163	Babu
40	(+)-Catechin 3-O-gallate	C₂₂H₁₈O₁₀	20.261	[M – H]⁻	442.0879	441.0806	441.0811	1.1	289, 169, 125	* Embu, Eeu
41	(+)-Catechin	C₁₅H₁₄O₆	20.437	** [M – H]⁻	290.0786	289.0713	289.0716	1.0	245, 205, 179	* Eeu, Dabu
42	Theaflavin 3,3′-O-digallate	C₄₃H₃₂O₂₀	24.533	[M – H]⁻	868.1448	867.1375	867.1373	−0.2	715, 563, 545	* Bmu, Beu, Bau
43	(−)-Epigallocatechin	C₁₅H₁₄O₇	30.714	** [M – H]⁻	306.0737	305.0664	305.0670	2.0	261, 219	* Aau, Dau, Deu, Debu, Eabu, Embu
Flavones
44	Apigenin 6-C-glucoside	C₂₁H₂₀O₁₀	16.981	[M – H]⁻	432.1077	431.1004	431.1015	2.6	413, 341, 311	* Dabu, Dmbu
45	Isorhamnetin	C₁₆H₁₂O₇	19.215	[M – H]⁻	316.0575	315.0502	315.0510	2.5	300, 271	Dmu
46	Apigenin 7-O-glucuronide	C₂₁H₁₈O₁₁	22.686	** [M – H]⁻	446.0875	445.0802	445.0802	0.2	271, 253	* Aau, Amu
47	3-Methoxysinensetin	C₂₁H₂₂O₈	30.813	[M – H]⁻	402.1301	401.1228	401.1234	1.5	388, 373, 355, 327	Eau
Flavanones
48	Hesperetin 3′,7-O-diglucuronide	C₂₈H₃₀O₁₈	4.806	[M – H]⁻	654.1424	653.1351	653.1356	0.8	477, 301, 286, 242	* Babu, Cmbu, Debu, Eabu
49	Narirutin	C₂₇H₃₂O₁₄	5.264	[M – H]⁻	580.1827	579.1754	579.1756	0.3	271	* Cmbu, Cabu, Cebu
50	Hesperetin 3′-sulfate	C₁₆H₁₄O₉S	7.389	[M – H]⁻	382.0354	381.0281	381.0277	−1.0	301, 286, 257, 242	* Aethbu, Ambu, Babu, Cmbu, Dmbu, Eabu
51	Hesperetin 3′-O-glucuronide	C₂₂H₂₂O₁₂	13.741	** [M – H]⁻	478.1130	477.1057	477.1062	1.0	301, 175, 113, 85	* Eau, Emu
52	Naringin 4′-O-glucoside	C₃₃H₄₂O₁₉	32.747	[M – H]⁻	742.2295	741.2222	741.2232	1.3	433, 271	Cethbu
Flavonols
53	Kaempferol 3-O-glucosyl-rhamnosyl-galactoside	C₃₃H₄₀O₂₀	4.719	[M – H]⁻	756.2135	755.2062	755.2075	1.7	285	Ceu
54	Quercetin 3′-O-glucuronide	C₂₁H₁₈O₁₃	5.185	[M – H]⁻	478.0758	477.0685	477.0679	−1.3	301	* Cabu, Bmu, Beu, Bethu, Bau
55	Patuletin 3-O-glucosyl-(1->6)-[apiosyl(1->2)]-glucoside	C₃₃H₄₀O₂₂	5.616	[M – H]⁻	788.1982	787.1909	787.1919	1.3	625, 463, 301, 271	* Ceu, Cau
56	Myricetin 3-O-arabinoside	C₂₀H₁₈O₁₂	7.182	[M – H]⁻	450.0821	449.0748	449.0750	0.4	317	* Cebu, Emu, Cau
57	Quercetin 3-O-glucosyl-xyloside	C₂₆H₂₈O₁₆	15.852	[M – H]⁻	596.1347	595.1274	595.1280	1.0	265, 138, 116 *	Eabu
58	Isorhamnetin 3-O-glucuronide	C₂₂H₂₀O₁₃	15.938	[M – H]⁻	492.0897	491.0824	491.0821	−0.6	315, 300, 272, 255	Aabu`
59	Myricetin 3-O-galactoside	C₂₁H₂₀O₁₃	16.921	** [M – H]⁻	480.0911	479.0838	479.0848	2.1	317	* Eeu, Deu, Beu, Bau
60	Myricetin 3-O-rhamnoside	C₂₁H₂₀O₁₂	17.690	[M – H]⁻	464.0937	463.0864	463.0879	3.2	317	* Eeu, Beu
61	Quercetin 3-O-arabinoside	C₂₀H₁₈O₁₁	20.258	** [M – H]⁻	434.0850	433.0777	433.0796	4.4	301	* Eeu, Dmu, Dmbu
62	6-Hydroxyluteolin 7-rhamnoside	C₂₁H₂₀O₁₁	20.989	[M – H]⁻	448.0991	447.0918	447.0904	−3.1	301	* Eeu, Eau
63	Quercetin 3′-sulfate	C₁₅H₁₀O₁₀S	31.909	[M – H]⁻	381.9980	380.9907	380.9905	−0.5	301	* Ceu, Cau, Beu, Bau, Aau
64	3-Methoxynobiletin	C₂₂H₂₄O₉	34.064	[M + H]⁺	432.1461	433.1534	433.1534	0.1	403, 385, 373, 345	* Aethu
Dihydroflavonols
65	Dihydromyricetin 3-O-rhamnoside	C₂₁H₂₂O₁₂	15.652	** [M – H]⁻	466.1112	465.1039	465.1043	0.9	301	* Eau, Deu
66	Dihydroquercetin	C₁₅H₁₂O₇	15.971	[M – H]⁻	304.0591	303.0518	303.0518	0.2	285, 275, 151	* Emu, Dabu
Dihydrochalcones
67	3-Hydroxyphloretin 2′-O-glucoside	C₂₁H₂₄O₁₁	4.593	[M – H]⁻	452.1354	451.1281	451.1280	−0.2	289, 273	Bau
Isoflavonoids
68	6″-O-Acetylglycitin	C₂₄H₂₄O₁₁	5.896	[M + H]⁺	488.1333	489.1406	489.1398	−1.6	285, 270	* Amu
69	2-Dehydro-O-desmethylangolensin	C₁₅H₁₂O₄	5.898	[M – H]⁻	256.0751	255.0678	255.0679	0.4	135, 119	* Cmbu, Babu
70	2′-Hydroxyformononetin	C₁₆H₁₂O₅	5.934	** [M – H]⁻	284.0687	283.0614	283.0619	1.8	270, 229	* Emu, Eeu, Dmu
71	Violanone	C₁₇H₁₆O₆	5.937	[M – H]⁻	316.0932	315.0859	315.0850	−2.9	300, 285, 135	* Cmbu, Embu, Eeu
72	Sativanone	C₁₇H₁₆O₅	16.707	** [M – H]⁻	300.0987	299.0914	299.0914	0.1	284, 269, 225	* Deu, Emu, Eau
73	Pseudobaptigenin	C₁₆H₁₀O₅	19.160	** [M – H]⁻	282.0506	281.0433	281.0431	−0.7	263, 237	* Dau, Aau
74	Dalbergin	C₁₆H₁₂O₄	21.505	[M – H]⁻	268.0717	267.0644	267.0656	4.5	252, 224, 180	* Deu, Ceu
75	6″-O-Malonyldaidzin	C₂₄H₂₂O₁₂	22.473	[M + H]⁺	502.1085	503.1158	503.1149	−1.8	255	Bau
76	Genistein 4′,7-O-diglucuronide	C₂₇H₂₆O₁₇	23.862	** [M – H]⁻	622.1174	621.1101	621.1122	3.4	269	Emu, Eeu, Dau, Cau, Bau, Aau
77	Glycitin	C₂₂H₂₂O₁₀	30.888	[M – H]⁻	446.1206	445.1133	445.1124	−2.0	285	* Cethbu, Eabu
Other polyphenols
Hydroxycoumarins
78	Esculetin	C₉H₆O₄	24.158	[M – H]⁻	178.0280	177.0207	177.0207	0.1	149, 133, 89	Bau
79	Scopoletin	C₁₀H₈O₄	31.117	[M – H]⁻	192.0419	191.0346	191.0347	0.5	176	* Bethbu, Eeu, Eau, Bmu, Beu, Bau
Hydroxybenzoketones
80	2-Hydroxy-4-methoxyacetophenone 5-sulfate	C₉H₁₀O₇S	24.081	** [M – H]⁻	262.0155	261.0082	261.0081	−0.4	181, 97	* Aeu, Aau
Phenolic terpenes
81	Carnosic acid	C₂₀H₂₈O₄	32.549	** [M – H]⁻	332.2004	331.1931	331.1933	0.6	287, 269	* Dethbu, Cethbu, Emu, Eau, Dmu, Deu, Dau, Ceu, Cethu, Cau, Bmu, Beu, Bethu, Bau, Amu, Aau
Tyrosols
82	3,4-DHPEA-AC	C₁₀H₁₂O₄	24.521	[M – H]⁻	196.0736	195.0663	195.0662	−0.5	135	Amu
83	3,4-DHPEA-EDA	C₁₇H₂₀O₆	29.370	[M – H]⁻	320.1270	319.1197	319.1192	−1.6	275, 195	* Aabu, Bmbu
Alkylmethoxyphenols
84	Equol	C₁₅H₁₄O₃	16.915	[M + H]⁺	242.0943	243.1016	243.1019	1.2	255, 211, 197	* Emu, Eau, Dau
Other polyphenols
85	Salvianolic acid C	C₂₆H₂₀O₁₀	16.686	[M – H]⁻	492.1026	491.0953	491.0976	4.7	311, 267, 249	Embu
86	Arbutin	C₁₂H₁₆O₇	19.621	[M – H]⁻	272.0901	271.0828	271.0827	−0.4	109	* Ceu, Cau
Lignans
87	Schisandrol B	C₂₃H₂₈O₇	3.062	** [M – H]⁻	416.1824	415.1751	415.1749	−0.5	224, 193, 165	Aebu
88	7-Hydroxymatairesinol	C₂₀H₂₂O₇	3.296	** [M – H]⁻	374.1381	373.1308	373.1311	0.8	343, 313, 298, 285	* Aebu, Eebu
89	Todolactol A	C₂₀H₂₄O₇	16.242	[M – H]⁻	376.1546	375.1473	375.1469	−1.1	313, 137	* Ambu, Dmbu
90	Sesamin	C₂₀H₁₈O₆	24.500	** [M – H]⁻	354.1138	353.1065	353.1068	0.8	338, 163	* Deu, Aabu, Babu
91	Arctigenin	C₂₁H₂₄O₆	26.476	[M – H]⁻	372.1565	371.1492	371.1494	0.5	356, 312, 295	* Embu, Eebu, Dmu, Dau, Cau, Aeu, Aau
92	Secoisolariciresinol-sesquilignan	C₃₀H₃₈O₁₀	31.907	[M – H]⁻	558.2434	557.2361	557.2387	4.7	539, 521, 509, 361	Amu
Stilbenes
93	Resveratrol	C₁₄H₁₂O₃	17.529	** [M – H]⁻	228.0787	227.0714	227.0717	1.3	212, 185, 157, 143	* Eau, Deu, Dabu
94	Resveratrol 5-O-glucoside	C₂₀H₂₂O₈	33.742	[M – H]⁻	390.1283	389.1210	389.1214	1.0	227	Debu

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* Compound was detected in more than one seaweed samples, data presented in this table are from asterisk sample. ** Compounds were detected in both negative [M – H]⁻ and positive [M + H]⁺ mode of ionization while only single mode data was presented. Seaweed samples were mentioned in abbreviations.

Table 5.

Characterization of phenolic compounds in conventional extracted seaweed samples by LC-ESI-QTOF-MS/MS.

No.	Proposed Compounds	Molecular Formula	RT (min)	Ionization (ESI⁺/ESI⁻)	Molecular Weight	Theoretical (m/z)	Observed (m/z)	Error (ppm)	MS2 Product Ions	Seaweed Samples
Phenolic acid
Hydroxybenzoic acids
1	Protocatechuic acid 4-O-glucoside	C₁₃H₁₆O₉	3.062	[M – H]⁻	316.0818	315.0745	315.0751	1.9	153	* Cethbc, Cebc, Debc, Dmbc
2	4-Hydroxybenzoic acid 4-O-glucoside	C₁₃H₁₆O₈	7.687	[M – H]⁻	300.0839	299.0766	299.0773	2.3	255, 137	Dabc
3	4-Hydroxybenzaldehyde	C₇H₆O₂	25.146	[M – H]⁻	122.0366	121.0293	121.0294	0.8	77	* Bethbc, Cmbc, Dabc, Dmbc
4	2,3-Dihydroxybenzoic acid	C₇H₆O₄	25.793	[M – H]⁻	154.0259	153.0186	153.0185	−0.7	109	Bethbc
5	2-Hydroxybenzoic acid	C₇H₆O₃	29.923	[M – H]⁻	138.0307	137.0234	137.0234	0.1	93	Bethbc
6	Gallic acid	C₇H₆O₅	31.949	[M – H]⁻	170.0209	169.0136	169.0140	2.4	125	* Aec, Aac, Bac, Bec, Bethc, Bmc, Cec, Cac, Cethc, Dec, Dethc, Dmc, Aabc, Debc
7	3-O-Methylgallic acid	C₈H₈O₅	33.658	[M – H]⁻	184.0355	183.0282	183.0280	−1.1	170, 142	* Dec, Dethc, Dmc
Hydroxycinnamic acids
8	Chlorogenic acid	C₁₆H₁₈O₉	3.078	[M – H]⁻	354.0969	353.0896	353.0902	1.7	253, 190, 144	* Cabc, Eabc
9	1-Sinapoyl-2,2′-diferuloylgentiobiose	C₄₃H₄₈O₂₁	3.119	[M – H]⁻	900.2677	899.2604	899.2574	−3.3	613, 201	* Aebc, Debc
10	3-Feruloylquinic acid	C₁₇H₂₀O₉	4.687	[M – H]⁻	368.1081	367.1008	367.1012	1.1	298, 288, 192, 191	Cmc
11	Ferulic acid 4-O-glucoside	C₁₆H₂₀O₉	4.821	[M – H]⁻	356.1100	355.1027	355.1031	1.1	193, 178, 149, 134	Eac
12	Feruloyl tartaric acid	C₁₄H₁₄O₉	4.958	[M – H]⁻	326.0641	325.0568	325.0562	−1.8	193, 149	* Dac, Dethc, Eac
13	Caffeic acid	C₉H₈O₄	5.116	[M – H]⁻	180.0431	179.0358	179.0359	0.6	143, 133	Emc
14	Cinnamic acid	C₉H₈O₂	7.400	** [M – H]⁻	148.0523	147.0450	147.0451	0.7	103	* Cmbc, Ambc
15	Caffeoyl glucose	C₁₅H₁₈O₉	7.457	[M – H]⁻	342.0922	341.0849	341.0837	−3.5	179, 161	Eebc
16	p-Coumaric acid 4-O-glucoside	C₁₅H₁₈O₈	15.736	[M – H]⁻	326.0993	325.0920	325.0920	0.1	163	Bebc
17	3-p-Coumaroylquinic acid	C₁₆H₁₈O₈	16.364	[M – H]⁻	338.0974	337.0901	337.0900	−0.3	265, 173, 162	Dabc
18	p-Coumaroyl tartaric acid	C₁₃H₁₂O₈	16.591	[M – H]⁻	296.0522	295.0449	295.0437	−4.1	115	Dmbc
19	Hydroxycaffeic acid	C₉H₈O₅	17.250	[M – H]⁻	196.0376	195.0303	195.0304	0.5	151	* Aabc, Bethbc, Dabc, Dethbc
20	Chicoric acid	C₂₂H₁₈O₁₂	17.787	[M – H]⁻	474.0826	473.0753	473.0739	−3.0	293, 311	Eac
21	Rosmarinic acid	C₁₈H₁₆O₈	18.986	[M – H]⁻	360.0823	359.0750	359.0753	0.8	179	* Dmbc, Eabc, emc
22	Cinnamoyl glucose	C₁₅H₁₈O₇	20.687	[M – H]⁻	310.1035	309.0962	309.0955	−2.3	147, 131, 103	* Dabc, Eabc
23	Sinapic acid	C₁₁H₁₂O₅	22.780	** [M – H]⁻	224.0674	223.0601	223.0598	−1.3	205, 163	* Cabc, Aabc, Ambc, Babc, Bebc, Bmbc, Cebc, Eabc, Eac
24	1,5-Dicaffeoylquinic acid	C₂₅H₂₄O₁₂	23.930	[M – H]⁻	516.1262	515.1189	515.1213	4.7	353, 335, 191, 179	Eec
25	Caffeic acid 3-O-glucuronide	C₁₅H₁₆O₁₀	24.346	** [M – H]⁻	356.0756	355.0683	355.0674	−2.5	179	* Bmc, Bac, Dmc, Ambc, Dethbc
26	1,2,2′-Triferuloylgentiobiose	C₄₂H₄₆O₂₀	30.541	[M – H]⁻	870.2536	869.2463	869.2486	2.6	693, 517	Aac
27	m-Coumaric acid	C₉H₈O₃	32.821	[M – H]⁻	164.0477	163.0404	163.0408	2.5	119	Eethc
28	Ferulic acid 4-O-glucuronide	C₁₆H₁₈O₁₀	33.763	[M + H]⁺	370.0902	371.0975	371.0974	−0.3	193	* Cebc, Dethbc
29	p-Coumaroyl malic acid	C₁₃H₁₂O₇	34.094	[M + H]⁺	280.0580	281.0653	281.0650	−1.1	163, 119	* Aebc, Ambc, Debc
Hydroxyphenylacetic acids
30	2-Hydroxy-2-phenylacetic acid	C₈H₈O₃	14.463	[M – H]⁻	152.0473	151.0400	151.0401	0.7	136, 92	* Cmbc, Aabc, Cabc, Dabc, Eabc, Eethc, Dmc, Eac
31	3,4-Dihydroxyphenylacetic acid	C₈H₈O₄	15.889	[M – H]⁻	168.0426	167.0353	167.0354	0.6	149, 123	* Aabc, Eethc
Hydroxyphenylpropanoic acids
32	Dihydroferulic acid 4-O-glucuronide	C₁₆H₂₀O₁₀	3.064	** [M – H]⁻	372.1073	371.1000	371.1004	1.1	195	* Aabc, Aebc, Cabc, Cebc, Cmbc, Debc, Dmbc, Eabc, Aethbc, Dethbc
33	Dihydrocaffeic acid 3-O-glucuronide	C₁₅H₁₈O₁₀	3.077	** [M – H]⁻	358.0905	357.0832	357.0833	0.3	181	* Aabc, Aebc, Eabc. Eebc, Aabc, Bac, Bec, Cmc
Flavonoids
Flavanols
34	(+)-Gallocatechin 3-O-gallate	C₂₂H₁₈O₁₁	7.625	[M – H]⁻	458.0830	457.0757	457.0755	−0.4	305, 169	* Babc, Bebc, Cebc, Debc
35	Prodelphinidin dimer B3	C₃₀H₂₆O₁₄	16.094	[M + H]⁺	610.1318	611.1391	611.1366	−4.1	469, 311, 291	Aebc
36	Theaflavin	C₂₉H₂₄O₁₂	16.799	[M – H]⁻	564.1257	563.1184	563.1190	1.1	545	* Dac, Emc, Embc
37	Procyanidin dimer B 1	C₃₀H₂₆O₁₂	18.209	** [M – H]⁻	578.1444	577.1371	577.1378	1.2	451	* Cabc, Aebc, Dethbc
38	(−)-Epigallocatechin	C₁₅H₁₄O₇	21.155	[M – H]⁻	306.0764	305.0691	305.0697	2.0	261, 219	* Aabc, Eabc
39	(+)-Catechin 3-O-gallate	C₂₂H₁₈O₁₀	24.152	[M – H]⁻	442.0876	441.0803	441.0816	2.9	289, 169, 125	Cac
40	Theaflavin 3,3′-O-digallate	C₄₃H₃₂O₂₀	26.891	[M – H]⁻	868.1512	867.1439	867.1439	0.1	715, 563, 545	Bac
41	4″-O-Methylepigallocatechin 3-O-gallate	C₂₃H₂₀O₁₁	28.257	[M – H]⁻	472.1005	471.0932	471.0923	−1.9	169, 319	* Cac, Bec, Debc
42	4′-O-Methyl-(−)-epigallocatechin 7-O-glucuronide	C₂₂H₂₄O₁₃	34.052	** [M – H]⁻	496.1186	495.1113	495.1117	0.8	451, 313	* Bmc, Bethc, Dmc, Cac, Dmc, Emc, Embc
Flavones
43	Apigenin 7-O-glucuronide	C₂₁H₁₈O₁₁	20.616	** [M + H]⁺	446.0826	445.0753	445.0755	0.4	271, 253	* Cac, Dmc, Emc, Aac, Dabc
44	Apigenin 7-O-(6″-malonyl-apiosyl-glucoside)	C₂₉H₃₀O₁₇	20.668	[M – H]⁻	650.1509	649.1436	649.1445	1.4	605	* Dmc, Aac
45	Apigenin 6-C-glucoside	C₂₁H₂₀O₁₀	21.777	[M – H]⁻	432.1045	431.0972	431.0980	1.9	413, 341, 311	* Dethc, Eec
46	Apigenin 7-O-apiosyl-glucoside	C₂₆H₂₈O₁₄	22.663	[M – H]⁻	564.1524	563.1451	563.1459	1.4	296	Eec
47	3-Methoxysinensetin	C₂₁H₂₂O₈	30.907	[M – H]⁻	402.1278	401.1205	401.1202	−0.7	388, 373, 355, 327	Dec
48	Apigenin 6,8-di-C-glucoside	C₂₇H₃₀O₁₅	32.223	[M – H]⁻	594.1592	593.1519	593.1509	−1.7	503, 473	* Bac, Aec, Bec, Bethc
Flavanones
49	Hesperetin 3′-sulfate	C₁₆H₁₄O₉S	7.732	[M – H]⁻	382.0358	381.0285	381.0283	−0.5	301, 286, 257, 242	* Babc, Bebc, Cebc, Dmbc, Embc
50	Hesperetin 3′-O-glucuronide	C₂₂H₂₂O₁₂	16.771	[M – H]⁻	478.1134	477.1061	477.1055	−1.3	301, 175, 113, 85	* Emc, Dac, Dmc, Dabc, Eabc, Eebc
51	Hesperetin 3′,7-O-diglucuronide	C₂₈H₃₀O₁₈	18.725	[M – H]⁻	654.1415	653.1342	653.1356	2.1	477, 301, 286, 242	Embc
52	Naringin 4′-O-glucoside	C₃₃H₄₂O₁₉	30.498	[M – H]⁻	742.2314	741.2241	741.2258	2.3	433, 271	Bec
53	Xanthohumol	C₂₁H₂₂O₅	31.272	[M – H]⁻	354.1486	353.1413	353.1425	3.4	338, 309	Aethc
Flavonols
54	Kaempferol 3-O-glucosyl-rhamnosyl-galactoside	C₃₃H₄₀O₂₀	4.718	[M – H]⁻	756.2092	755.2019	755.2012	−0.9	285	Cmc
55	Kaempferol 3,7-O-diglucoside	C₂₇H₃₀O₁₆	16.377	[M – H]⁻	610.1542	609.1469	609.1474	0.8	447, 285	Amc
56	Isorhamnetin 3-O-glucuronide	C₂₂H₂₀O₁₃	16.474	[M – H]⁻	492.0928	491.0855	491.0872	3.5	315, 300, 272, 255	* Dac, Dmc
57	Myricetin 3-O-rhamnoside	C₂₁H₂₀O₁₂	17.258	[M – H]⁻	464.0946	463.0873	463.0867	−1.3	317	* Dmc, Bac, Bec, Dmc, Eec, Emc
58	Quercetin 3-O-arabinoside	C₂₀H₁₈O₁₁	18.213	[M – H]⁻	434.0837	433.0764	433.0772	1.8	301	* Dac, Dmc
59	Myricetin 3-O-arabinoside	C₂₀H₁₈O₁₂	18.941	[M – H]⁻	450.0825	449.0752	449.0741	−2.4	317	Dmc
60	6-Hydroxyluteolin 7-rhamnoside	C₂₁H₂₀O₁₁	19.191	[M – H]⁻	448.1003	447.0930	447.0933	0.7	301	* Emc, Cac, Dac, Dethc, Eec, Emc, Cmbc, Eebc
61	Quercetin 3-O-(6”-malonyl-glucoside)	C₂₄H₂₂O₁₅	23.143	[M – H]⁻	550.0977	549.0904	549.0886	−3.3	303	Bac
62	Myricetin 3-O-galactoside	C₂₁H₂₀O₁₃	24.356	[M – H]⁻	480.0909	479.0836	479.0837	0.2	317	* Emc, Bac, Bethc, Dmc
63	Quercetin 3-O-glucosyl-xyloside	C₂₆H₂₈O₁₆	25.144	[M – H]⁻	596.1402	595.1329	595.1313	−2.7	265, 138, 116	Bac
64	Quercetin 3′-O-glucuronide	C₂₁H₁₈O₁₃	31.165	[M – H]⁻	478.0785	477.0712	477.0717	1.0	301	* Bec, Bac
65	Quercetin 3′-sulfate	C₁₅H₁₀O₁₀S	32.872	[M – H]⁻	382.0021	380.9948	380.9951	0.8	301	* Bmc, Aec, Bethc, Dec, Eac
66	Quercetin 3-O-xylosyl-rutinoside	C₃₂H₃₈O₂₀	33.752	[M + H]⁻	742.1933	743.2006	743.2003	−0.4	479, 317	Debc
Dihydroflavonols
67	Dihydroquercetin 3-O-rhamnoside	C₂₁H₂₂O₁₁	19.734	[M – H]⁻	450.1147	449.1074	449.1077	0.7	303	* Eec, Dethc, Dmc, Emc, Eabc, Dabc, Eabc
68	Dihydromyricetin 3-O-rhamnoside	C₂₁H₂₂O₁₂	23.443	[M – H]⁻	466.1111	465.1038	465.1047	1.9	301	* Emc, Dac, Dethc, Eec
Dihydrochalcones
69	3-Hydroxyphloretin 2′-O-glucoside	C₂₁H₂₄O₁₁	4.663	[M – H]⁻	452.1348	451.1275	451.1277	0.4	289, 273	* Aac, Aec, Bec, Cac, Eec
Anthocyanins
70	Delphinidin 3-O-glucoside	C₂₁H₂₁O₁₂	26.489	[M – H]⁻	465.1016	464.0943	464.0940	−0.6	303	Bac
71	Pelargonidin	C₁₅H₁₁O₅	32.266	[M – H]⁻	271.0618	270.0545	270.0557	4.4	243, 197, 169, 141	Eac
72	Cyanidin 3,5-O-diglucoside	C₂₇H₃₁O₁₆	32.278	[M + H]⁺	611.1632	612.1705	612.1711	1.0	449, 287	* Aethbc, Bebc, Debc
73	Peonidin 3-O-diglucoside-5-O-glucoside	C₃₄H₄₃O₂₁	33.383	[M – H]⁻	787.2331	786.2258	786.2246	−1.5	625, 478, 317	* Cethc, Dec
Isoflavonoids
74	6″-O-Acetyldaidzin	C₂₃H₂₂O₁₀	4.703	[M – H]⁻	458.1218	457.1145	457.1139	−1.3	221	Bmc
75	5,6,7,3′,4′-Pentahydroxyisoflavone	C₁₅H₁₀O₇	5.266	[M – H]⁻	302.0439	301.0366	301.0376	3.3	285, 257	Amc
76	2′,7-Dihydroxy-4′,5′-dimethoxyisoflavone	C₁₇H₁₄O₆	6.882	[M – H]⁻	314.0767	313.0694	313.0698	1.3	300, 282	* Eac, Eec
77	6″-O-Malonylgenistin	C₂₄H₂₂O₁₃	14.363	** [M + H]⁺	518.1069	517.0996	517.0997	0.2	271	* Eac, Eac
78	2-Dehydro-O-desmethylangolensin	C₁₅H₁₂O₄	16.548	[M – H]⁻	256.0746	255.0673	255.0667	−2.4	135, 119	* Emc, Bmc
79	Sativanone	C₁₇H₁₆O₅	16.563	** [M – H]⁻	300.0994	299.0921	299.0923	0.7	284, 269, 225	* Aec, Aac, Amc, Bec, Cac, Emc, Ambc
80	6″-O-Malonylglycitin	C₂₅H₂₄O₁₃	18.919	[M – H]⁻	532.1215	531.1142	531.1145	0.6	285, 270, 253	* Emc, Eec
81	Formononetin 7-O-glucuronide	C₂₂H₂₀O₁₀	21.977	[M – H]⁻	444.1048	443.0975	443.0972	−0.7	267, 252	* Cac, Eac
82	2′-Hydroxyformononetin	C₁₆H₁₂O₅	22.421	[M + H]⁺	284.0694	285.0767	285.0764	−1.1	270, 229	* Dmc, Cac, Emc
83	Violanone	C₁₇H₁₆O₆	24.296	[M – H]⁻	316.0921	315.0848	315.0842	−1.9	300, 285, 135	* Aebc, Ambc
84	Genistein 4′,7-O-diglucuronide	C₂₇H₂₆O₁₇	25.445	[M – H]⁻	622.1114	621.1041	621.1067	4.2	269	* Aac, Bec, Dethc, Debc
85	6″-O-Malonyldaidzin	C₂₄H₂₂O₁₂	33.752	** [M + H]⁺	502.1093	503.1166	503.1178	2.4	255	* Debc, Dabc, Embc, Dac, Cac, Eac, Emc
Other polyphenols
Hydroxycoumarins
86	Esculetin	C₉H₆O₄	24.191	[M – H]⁻	178.0248	177.0175	177.0171	−2.3	149, 133, 89	Bec
87	Scopoletin	C₁₀H₈O₄	31.153	[M – H]⁻	192.0407	191.0334	191.0335	0.5	176	* Babc, Aebc, Bebc, Bethbc, Bmbc, Dabc, Debc, Dmbc, Dmc, Bec, Bmc, Cac, Eec, Emc
Hydroxybenzaldehydes
88	p-Anisaldehyde	C₈H₈O₂	30.932	[M – H]⁻	136.0516	135.0443	135.0442	−0.7	122, 109	* Bethc, Eac, Eec, Eethc, Emc
89	3-Hydroxy-3-(3-hydroxyphenyl) propionic acid	C₉H₁₀O₄	32.498	[M – H]⁻	182.0582	181.0509	181.0507	−1.1	163, 135, 119	Eac
Phenolic terpenes
90	Carnosic acid	C₂₀H₂₈O₄	32.545	[M – H]⁻	332.1985	331.1912	331.1906	−1.8	287, 269	* Bac, Aac, Aec, Aethc, Amc, Bac, Bec, Bethc, Bmc, Cec, Dac, Dethc, Eec, Emc, Dethbc, Eabc
Tyrosols
91	3,4-DHPEA-AC	C₁₀H₁₂O₄	4.682	[M – H]⁻	196.0727	195.0654	195.0654	0.1	135	Cac
92	Hydroxytyrosol 4-O-glucoside	C₁₄H₂₀O₈	16.175	[M – H]⁻	316.1149	315.1076	315.1076	0.1	153, 123	Cmbc
93	3,4-DHPEA-EDA	C₁₇H₂₀O₆	28.718	[M – H]⁻	320.1280	319.1207	319.1209	0.6	275, 195	Bethbc
Alkylmethoxyphenols
94	Equol	C₁₅H₁₄O₃	18.110	** [M + H]⁺	242.0943	243.1016	243.1014	−0.8	255, 211, 197	* Cabc, Amc, Aac, Aec, Eac, Eethc
Other polyphenols
95	Salvianolic acid B	C₃₆H₃₀O₁₆	16.693	[M – H]⁻	718.1517	717.1444	717.1421	−3.2	519, 339, 321, 295	Embc
96	Lithospermic acid	C₂₇H₂₂O₁₂	17.324	** [M – H]⁻	538.1097	537.1024	537.1018	−1.1	493, 339, 295	* Eabc, Cabc, Cethbc, Dmc, Cec
97	Salvianolic acid C	C₂₆H₂₀O₁₀	21.929	[M – H]⁻	492.1052	491.0979	491.0989	2.0	311, 267, 249	* Debc, Dmbc
Lignans
98	Todolactol A	C₂₀H₂₄O₇	16.368	[M – H]⁻	376.1539	375.1466	375.1470	1.1	313, 137	* Debc, Embc
99	7-Hydroxymatairesinol	C₂₀H₂₂O₇	28.718	** [M – H]⁻	374.1379	373.1306	373.1311	1.3	343, 313, 298, 285	* Bethbc, Dabc, Eebc, aac
100	Arctigenin	C₂₁H₂₄O₆	30.493	[M – H]⁻	372.1565	371.1492	371.1475	−4.6	356, 312, 295	Aethc
101	Secoisolariciresinol-sesquilignan	C₃₀H₃₈O₁₀	30.674	[M – H]⁻	558.2463	557.2390	557.2390	0.1	539, 521, 509, 361	Aec, debc
102	7-Oxomatairesinol	C₂₀H₂₀O₇	32.347	[M + H]⁺	372.1184	373.1257	373.1256	−0.3	358, 343, 328, 325	* Babc, Bebc, Bethbc, Cmbc
103	Sesamin	C₂₀H₁₈O₆	32.442	[M – H]⁻	354.1124	353.1051	353.1061	2.8	338, 163	Eec
Stilbenes
104	Resveratrol 5-O-glucoside	C₂₀H₂₂O₈	16.382	[M – H]⁻	390.1310	389.1237	389.1234	−0.8	227	Embc

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Gallic acid ([M – H]⁻, m/z 169.0154), 2-hydroxybenzoic acid ([M – H]⁻, m/z 137.0238) and 2,3-dihydroxybenzoic acid ([M – H]⁻, m/z 153.0190) were identified at product ions m/z 125, m/z 93 and at m/z 109 due to the corresponding loss of CO₂ [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 CH₃, CO₂, and CH₃ with CO₂ 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 CO₂ (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].

Quercetin 3′-O-glucuronide ([M – H]⁻ m/z at 477.0679) had product ion at m/z 301 in the MS² spectrum due to the loss of glucuronide (176 Da) from the precursor [73]. Myricetin 3-O-arabinoside ([M – H]⁻ m/z at 449.0750) had peaks at m/z 317 (loss of pentose moiety, 132 Da) which confirmed the identity of myricetin 3-O-arabinoside [74]. Quercetin 3′-O-glucuronide was identified in samples ultrasonication methodology of bound form in Durvillaea sp. (70% acetone), and free form in Ecklonia radiata (70% methanol, 70% ethanol, 70% acetone, absolute ethyl acetate). Myricetin 3-O-arabinoside in Durvillaea sp. (70% ethanol and 70% acetone extract) and Cystophora sp. (70% methanol extract). The compounds quercetin 3′-O-glucuronide and myricetin 3-O-arabinoside were identified in free form in conventional methodology in seaweed samples of Ecklonia radiata (70% ethanol and 70% acetone extract). The biosynthesis of quercetin via hydroxylation reaction of dihydrokaempferol forms dihydroquercetin in the presence of the enzyme flavonol 3′-hydroxylase. In the following step of biosynthesis, dihydroquercetin catalyzes in the presence of enzyme flavonol synthase to form quercetin [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].

Sativanone was identified in both modes of ionization and tentatively identified by the precursor ions at m/z 299.0914 and the product ions at m/z 284 (M – H – 15, loss of CH₃ from B-ring) and at m/z 269 (M – H – 30, loss of two CH₃) and at m/z 225 (M – H – 74, loss of two CH₃ and CO₂) [78]. It was identified by the samples Sargassum sp. (70% ethanol extract) and Cystophora sp. (70% methanol, 70% ethanol extract) in their free form by ultrasonication methodology while Phyllospora comosa (70% ethanol, 70% acetone, 70% methanol extract), Ecklonia radiata (70% ethanol extract), Durvillaea sp. (70% acetone extract) and Cystophora sp. (70% methanol extract), by conventional methodology in free and bound forms. Previously, restharrow root has been used in traditional medicine identified sativanone [79]. Ethanolic extracts of Dalbergia odorifera-treated mice when exposed to UVB significantly reduced ROS levels and the number of senescent cells in the skin [80]. Dalbergin compound was tentatively identified with [M – H]⁻ m/z at 267.0656 exhibited characteristic fragment ions at m/z 252 [M – H – CH₃], m/z 224 [M – H – CH₃ – CO] and m/z 180 [M – H – CH₃ – CO – CO₂] [78]. It was identified in Durvillaea sp., Sargassum sp. in both ultrasonication and conventional methodologies. Dalbergin was first isolated from Dalbergia odorifera [81]. Dalbergin is widely used traditionally as an anti-inflammatory, anti-pyretic, analgesic, antioxidant, anti-diabetic, antimicrobial, and anti-cancer agent [82].

Scopoletin ([M – H]⁻ m/z at 191.0347) was identified by the product ions at m/z 176 [M – H – CH₃] and m/z 147 [M – H – CO₂] [83]. It was identified in Ecklonia radiata (absolute ethyl acetate, 70% ethanol, 70% methanol, 70% acetone) and Cystophora sp. (70% ethanol and 70% acetone extract) in ultrasonication methodology whereas in conventional, it was identified in Ecklonia radiata (70% ethanol, 70% methanol extract), Durvillaea sp. (70% acetone), Sargassum sp. (70% ethanol, 70% methanol) and Cystophora sp. (70% ethanol, 70% methanol). This compound was previously detected in seaweeds, including Codium sp. (Chlorophyta), Grateloupia sp. (Rhodophyta), and Sargassum sp., (Phaeophyceae) [20]. Scopoletin was also detected in curry plant, cassava, candlenut tree, giant potato, sweet wormwood, chinaberry tree, sugar maple, perfume flower tree, and white mulberry [84]. Scopoletin has been shown to possess antimicrobial properties, reduce inflammations, and decrease cardiovascular diseases [85]. Scopoletin extracted from the Hypochaeris radicata demonstrated anti-inflammatory and antioxidant properties by suppressing the production of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 [86].

Resveratrol ([M – H]⁻ m/z at 227.0717) was detected in both ionization modes. Resveratrol observed the fragmentation ions at m/z 212 [M – H – CH₃], m/z 185 [M – H – CHCOH], m/z 157 [M – H – CHCOH – CO], and m/z 143 [M – H – CHCOH – C₂H₂O] [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].

2.6. 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.

(A) Heatmap showing phenolic compounds’ distribution and concentration among five seaweed species extracted in four different solvents via ultrasonication extraction. (B) Heatmap showing phenolic compounds’ distribution and concentration among five seaweed species extracted in four different solvents via conventional extraction.

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.

3. Materials and Methods

3.1. Chemicals

The chemicals used for extraction were methanol, ethanol, ethyl acetate, acetone, and formic acid of analytical grade. For the in vitro assays, the standards used were gallic acid, phloroglucinol, ethylenediaminetetraacetic acid (EDTA), quercetin, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), and catechin, obtained from Sigma Aldrich (St. Louis, MO, USA). The chemicals used for estimation of phenolic and antioxidant potential were 2,2′-diphenyl-1-picrylhydrazl (DPPH), 2,4,6-tripyridyl-s-triazine (TPTZ), Folin–Ciocalteu’s phenol reagent, vanillin, aluminium chloride hexahydrate, ferric (III) chloride anhydrous, potassium persulfate, 2-2′-azino-bis(3-ethylbenz-thiazoline-6-sulphonate) (ABTS), 3-ethylbenzothiazoline-6-sulphonic acid, sodium phosphate dibasic heptahydrate, iron (II) chloride, sodium phosphate monobasic monohydrate, iron (II) sulfate heptahydrate, 3-hydroxybenzoic acid, ferrozine, potassium ferricyanide, 2,4-dimethoxybenzaldehyde (DMBA), ferric ammonium sulfate, potassium ferricyanide, sodium tungstate, absolute ethanol and dodeca-molybdophosphoric acid, purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Methanol, sodium hydroxide pellets, sodium carbonate anhydrous, and hydrogen peroxide (30%) were purchased from Chem-Supply Pty Ltd. (Adelaide, SA, Australia). Ethyl acetate, sodium acetate (hydrated), formic acid 99%, and acetone were purchased from Chem-Supply Pvt Ltd. and Ajax Finecham, respectively (VIC, Melbourne, Australia). Glacial acetic acid was purchased from Thermo Fisher Scientific Inc (Waltham, MA, USA). Milli-Q water by Millipore Milli-Q Gradient Water Purification System (Darmstadt, Germany). The 98% sulphuric acid was procured from RCI Labscan Ltd. (Bangkok, Thailand).

3.2. 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.

Seaweed samples used in our study, Sample (A) *Phyllospora comosa*, Sample (B) *Ecklonia radiata*, Sample (C) *Durvillaea* sp., Sample (D) *Sargassum* sp., Sample (E) *Cystophora* sp.

3.3. 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.

3.4. Extraction Preparation

3.4.1. Free Phenolics Extraction

The conventional and ultrasonication of free phenolics were performed and slightly modified as described by Čagalj 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.

3.4.2. 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 10 mL of respective solvents. The samples were centrifuged for 15 min at 5000 rpm under 4 °C using Hettich Refrigerated Centrifuge (ROTINA380R, Tuttlingen, BadenWürttemberg, Germany). The supernatant fluid was filtered via 0.45 µm syringe filter (Thermo Fisher Scientific Inc., Waltham, MA, USA) and collected as bound phenolic extracts.

3.5. 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.

3.5.1. 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).

3.5.2. 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.).

3.5.3. 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.).

3.5.4. 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).

3.5.5. 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 FeNH₄(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 K₃Fe(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 (Na₂WO₄·2H₂O) and 5 g of dodeca-molybdophosphoric acid (12MoO₃·H₃PO₄·H₂O) 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).

3.5.7. 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.

3.5.8. 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.

3.5.10. 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).

3.5.12. 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.

3.5.13. 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.

3.6. 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).

3.7. 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 0.45 μm syringe filter (PVDF, Millipore, MA, USA). A Synergi Hydro-RP (250 × 4.6 mm i.d.) reversed-phase column with a particle size of 4 µm (Phenomenex, Lane Cove, NSW, Australia) was protected by a Phenomenex 4.0 × 2.0 mm i.d., C18 ODS guard column. The injection volume of the sample or standard was 25 μL. The mobile phase A and B were of water/acetic acid (98:2, v/v) and acetonitrile/water/acetic acid (50:50:2, v/v/v), respectively. The gradient profile was 90–10% B (0–20 min), 75–30% B (20–30 min), 65–35% B (30–40 min), 45–55% B (40–60 min), 90–10% B (60–61 min), 90–10% B (61–66 min). 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.

3.8. 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.

4. 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.

Acknowledgments

We would like to thank James Redmond for helping us with the sample collection. I also would like to extend my gratitude to Nicholas Williamson, Shuai Nie, and Michael Leeming from the Mass Spectrometry and Proteomics Facility, Bio21 Molecular Science and Biotechnology Institute, the University of Melbourne, VIC, Australia for providing access and support for the use of LC-ESI-QTOF-MS/MS and data analysis. We would like to express our gratitude to the Turkish government’s TUBITAK 2219 fellowship programme for their support.

Abbreviations

Amu: Phyllospora comosa methanol ultrasound extract; Aeu: Phyllospora comosa ethanol ultrasound extract; Aau: Phyllospora comosa acetone ultrasound extract; Aethu: Phyllospora comosa ethyl acetate ultrasound extract; Bmu: Ecklonia radiata methanol ultrasound extract; Beu: Ecklonia radiata ethanol ultrasound extract; Bau: Ecklonia radiata acetone ultrasound extract; Bethu: Ecklonia radiata ethyl acetate ultrasound extract; Cmu: Durvillaea sp., methanol ultrasound extract; Ceu: Durvillaea sp., ethanol ultrasound extract; Cau: Durvillaea sp., acetone ultrasound extract; cethu: Durvillaea sp., ethyl acetate ultrasound extract; Dmu: Sargassum sp., methanol ultrasound extract; Deu: Sargassum sp., ethanol ultrasound extract; Dau: Sargassum sp., acetone ultrasound extract; Dethu: Sargassum sp., ethyl acetate ultrasound extract; Emu: Cystophora sp., methanol ultrasound extract; Eeu: Cystophora sp., ethanol ultrasound extract; Eau: Cystophora sp., acetone ultrasound extract; Eethu: Cystophora sp., ethyl acetate ultrasound extract; Ambu: Phyllospora comosa methanol bound ultrasound extract; Aebu: Phyllospora comosa ethanol bound ultrasound extract; Aabu: Phyllospora comosa acetone bound ultrasound extract; Aethbu: Phyllospora comosa ethyl acetate bound ultrasound extract; Bmbu: Ecklonia radiata methanol bound ultrasound extract; Bebu: Ecklonia radiata ethanol bound ultrasound extract; Babu: Ecklonia radiata acetone bound ultrasound extract; Bethbu: Ecklonia radiata ethyl acetate bound ultrasound extract; Cmbu: Durvillaea sp., methanol bound ultrasound extract; Cebu: Durvillaea sp., ethanol bound ultrasound extract; Cabu: Durvillaea sp., acetone bound ultrasound extract; Cethbu: Durvillaea sp., ethyl acetate bound ultrasound extract; Dmbu: Sargassum sp., methanol bound ultrasound extract; Debu: Sargassum sp., ethanol bound ultrasound extract; Dabu: Sargassum sp., acetone bound ultrasound extract; Dethbu: Sargassum sp., ethyl acetate bound ultrasound extract; Embu: Cystophora sp., methanol bound ultrasound extract; Eebu: Cystophora sp., ethanol bound ultrasound extract; Eabu: Cystophora sp., acetone bound ultrasound extract; Eethbu: Cystophora sp., ethyl acetate bound ultrasound extract.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ph16050773/s1, Table S1: Estimation of freeze-dried free phenolics of seaweed species extracted by the conventional and non-conventional method; Table S2: Estimation of freeze-dried bound phenolics of seaweed species extracted by the conventional and non-conventional method; Table S3: Estimation of freeze-dried free phenolic’s antioxidant potential of seaweed species extracted by the conventional and non-conventional method; Table S4: Estimation of freeze-dried bound antioxidant potential of seaweed species extracted by the conventional and non-conventional method; Table S5: Characterization of free phenolic compounds in different seaweed samples with different extraction methods by LC-ESI-QTOF-MS/MS; Table S6: Characterization of bound phenolic compounds in different seaweed samples with different extraction methods by LC-ESI-QTOF-MS/MS.

Click here for additional data file.^{(278.8KB, zip)}

Author Contributions

Conceptualization, methodology, formal analysis, validation and investigation, V.S. and H.A.R.S.; resources, H.A.R.S., C.J.B. and F.R.D.; writing—original draft preparation, V.S. and H.A.R.S.; writing—review and editing, V.S., F.E., O.T.A., C.J.B., H.A.R.S. and F.R.D.; supervision, H.A.R.S. and C.J.B.; ideas sharing, H.A.R.S.; C.J.B. and F.R.D.; funding acquisition, H.A.R.S., F.R.D. and C.J.B. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in this article.

Conflicts of Interest

The authors declare no conflict of interest.

Funding Statement

This research was funded by the Deakin university under “Deakin University Postgraduate Research Scholarship (DUPRS) scheme”, Deakin DVCR-funded scholarship supporting Deakin BioFactory research; “Collaborative Research Development Grant” (Grant No. UoM-21/23) funded by the University of Melbourne and “Australian Research Council—Discovery Early Career Award” (ARC-DECRA—DE220100055) funded by the Australian Government.

Footnotes

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

Vigasini Subbiah

Faezeh Ebrahimi

Osman T Agar

Frank R Dunshea

Colin J Barrow

Hafiz A R Suleria

Roles

Abstract

1. Introduction

2. Results and Discussion

2.1. Total Phenolics, Flavonoids Tannin and Phlorotannin Content

Table 1.

2.2. Antioxidant Potential

Table 2.

2.3. Correlation

Table 3.

Figure 1.

2.4. Distribution of Phenolic Compounds in Seaweeds

Figure 2.

Figure 3.

Figure 4.

Figure 5.

2.5. LC-ESI-QTOF-MS/MS Characterization of Phenolic Compounds

Table 4.

Table 5.

2.6. Heatmap Analysis of Quantified Phenolics in Seaweeds

Figure 6.

3. Materials and Methods

3.1. Chemicals

3.2. Seaweed Collection and Identification of Seaweed Samples

Figure 7.

3.3. Sample Preparation

3.4. Extraction Preparation

3.4.1. Free Phenolics Extraction

3.4.2. Bound Phenolic Extraction

3.5. Estimation of Phenolic and Antioxidant Assays

3.5.1. Estimation of Total Phenolic Compound

3.5.2. Determination of Total Flavonoid Compounds (TFC)

3.5.3. Determination of Total Tannin Content (TTC)

3.5.4. 2,4-Dimethoxybenzaldehyde Assay (DMBA)

3.5.5. Prussian Blue Assay (PBA)

3.5.6. Folin–Denis Assay (FDA)

3.5.7. 2,2′-Diphenyl-1-Picrylhydrazyl (DPPH) Assay

3.5.8. Ferric Reducing Antioxidant Power (FRAP) Assay

3.5.9. 2,2′-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid (ABTS) Assay

3.5.10. Estimation of Hydroxyl Radical Scavenging Activity (OH-RSA)

3.5.11. Estimation of Ferrous Ion Chelating Activity (FICA)

3.5.12. Estimation of Reducing Power (RPA)

3.5.13. Total Antioxidant Capacity (TAC)

3.6. Characterization of Phenolic Compounds by LC-ESI-QTOF-MS/MS Analysis

3.7. HPLC-PDA Analysis

3.8. Statistical Analysis

4. Conclusions

Acknowledgments

Abbreviations

Supplementary Materials

Author Contributions

Institutional Review Board Statement

Informed Consent Statement

Data Availability Statement

Conflicts of Interest

Funding Statement

Footnotes

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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