Next Article in Journal
Rutin Gel with Bone Graft Accelerates Bone Formation in a Rabbit Model by Inhibiting MMPs and Enhancing Collagen Activities
Next Article in Special Issue
Structure–Activity Relationship Studies of Indolglyoxyl-Polyamine Conjugates as Antimicrobials and Antibiotic Potentiators
Previous Article in Journal
Prescribed Drugs and Self-Directed Violence: A Descriptive Study in the Spanish Pharmacovigilance Database
Previous Article in Special Issue
Exploring the Benefits of Phycocyanin: From Spirulina Cultivation to Its Widespread Applications
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Pharmaceuticals
Volume 16
Issue 5
10.3390/ph16050773
pharmaceuticals-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

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

by
Vigasini Subbiah
1,2,
Faezeh Ebrahimi
2,
Osman T. Agar
2,
Frank R. Dunshea
2,3,
Colin J. Barrow
1 and
Hafiz A. R. Suleria
1,2,*
1
Centre for Sustainable Bioproducts, Deakin University, Waurn Ponds, VIC 3217, Australia
2
School of Agriculture, Food and Ecosystem Sciences, Faculty of Science, The University of Melbourne, Parkville, VIC 3010, Australia
3
Faculty of Biological Sciences, The University of Leeds, Leeds LS2 9JKT, UK
*
Author to whom correspondence should be addressed.
Pharmaceuticals 2023, 16(5), 773; https://doi.org/10.3390/ph16050773
Submission received: 27 April 2023 / Revised: 17 May 2023 / Accepted: 18 May 2023 / Published: 22 May 2023
(This article belongs to the Special Issue Plant- , Microbial- and Marine-Derived Natural Product Research in Drug Discovery: Strengths and Perspectives)
Browse Figures
Versions Notes

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.

Graphical Abstract

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).
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].
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.
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).

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.
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.
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].
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.

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.
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.
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.

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.

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.

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.

Funding

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.

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.

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.

Conflicts of Interest

The authors declare no conflict of interest.

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.

References

  1. Palmieri, N.; Forleo, M.B. The potential of edible seaweed within the western diet. A segmentation of italian consumers. Int. J. Gastron. Food Sci. 2020, 20, 100202. [Google Scholar] [CrossRef]
  2. Pereira, L. A Review of the Nutrient Composition of Selected Edible Seaweeds; Nova Science Publishers, Inc.: Hauppauge, NY, USA, 2011; Volume 7, pp. 15–47. [Google Scholar]
  3. Rajapakse, N.; Kim, S.K. Nutritional and digestive health benefits of seaweed. Adv. Food Nutr. Res. 2011, 64, 17–28. [Google Scholar] [PubMed]
  4. Al Jitan, S.; Alkhoori, S.A.; Yousef, L.F. Chapter 13—Phenolic acids from plants: Extraction and application to human health. In Studies in Natural Products Chemistry; Atta-ur, R., Ed.; Elsevier: Amsterdam, The Netherlands, 2018; Volume 58, pp. 389–417. [Google Scholar]
  5. Cotas, J.; Pacheco, D.; Gonçalves, A.M.M.; Silva, P.; Carvalho, L.G.; Pereira, L. Seaweeds’ nutraceutical and biomedical potential in cancer therapy: A concise review. J. Cancer Metastasis Treat. 2021, 2021, 13. [Google Scholar] [CrossRef]
  6. Harb, T.B.; Chow, F. An overview of beach-cast seaweeds: Potential and opportunities for the valorization of underused waste biomass. Algal Res. 2022, 62, 102643. [Google Scholar] [CrossRef]
  7. Chan, J.C.C.; Cheung, P.C.K.; Ang, P.O. Comparative studies on the effect of three drying methods on the nutritional composition of seaweed Sargassum hemiphyllum (turn.) c. Ag. J. Agric. Food Chem. 1997, 45, 3056–3059. [Google Scholar] [CrossRef]
  8. Ciurzyńska, A.; Lenart, A. Freeze-drying—Application in food processing and biotechnology—A review. Pol. J. Food Nutr. Sci. 2011, 61, 165–171. [Google Scholar] [CrossRef]
  9. Cruces, E.; Rojas-Lillo, Y.; Ramirez-Kushel, E.; Atala, E.; López-Alarcón, C.; Lissi, E.; Gómez, I. Comparison of different techniques for the preservation and extraction of phlorotannins in the kelp Lessonia spicata (phaeophyceae): Assays of dpph, orac-pgr, and orac-fl as testing methods. J. Appl. Phycol. 2015, 28, 573–580. [Google Scholar] [CrossRef]
  10. Turkmen, N.; Sari, F.; Velioglu, Y.S. Effects of extraction solvents on concentration and antioxidant activity of black and black mate tea polyphenols determined by ferrous tartrate and folin–ciocalteu methods. Food Chem. 2006, 99, 835–841. [Google Scholar] [CrossRef]
  11. Yingngam, B.; Monschein, M.; Brantner, A. Ultrasound-assisted extraction of phenolic compounds from Cratoxylum formosum ssp. Formosum leaves using central composite design and evaluation of its protective ability against H2O2-induced cell death. Asian Pac. J. Trop. Med. 2014, 7S1, S497–S505. [Google Scholar] [CrossRef]
  12. Rostagno, M.A.; Prado, J.M. Natural Product Extraction: Principles and Applications; Royal Society of Chemistry: London, UK, 2013. [Google Scholar]
  13. Rodrigues, D.; Sousa, S.; Silva, A.; Amorim, M.; Pereira, L.; Rocha-Santos, T.A.; Gomes, A.M.; Duarte, A.C.; Freitas, A.C. Impact of enzyme- and ultrasound-assisted extraction methods on biological properties of red, brown, and green seaweeds from the central west coast of portugal. J. Agric. Food Chem. 2015, 63, 3177–3188. [Google Scholar] [CrossRef]
  14. Kadam, S.U.; Tiwari, B.K.; O’Donnell, C.P. Effect of ultrasound pre-treatment on the drying kinetics of brown seaweed Ascophyllum nodosum. Ultrason. Sonochem. 2015, 23, 302–307. [Google Scholar] [CrossRef] [PubMed]
  15. Hassan, I.H.; Pham, H.N.T.; Nguyen, T.H. Optimization of ultrasound-assisted extraction conditions for phenolics, antioxidant, and tyrosinase inhibitory activities of vietnamese brown seaweed (Padina australis). J. Food Process. Preserv. 2021, 45, e15386. [Google Scholar] [CrossRef]
  16. Ummat, V.; Tiwari, B.K.; Jaiswal, A.K.; Condon, K.; Garcia-Vaquero, M.; O’Doherty, J.; O’Donnell, C.; Rajauria, G. Optimisation of ultrasound frequency, extraction time and solvent for the recovery of polyphenols, phlorotannins and associated antioxidant activity from brown seaweeds. Mar. Drugs 2020, 18, 250. [Google Scholar] [CrossRef] [PubMed]
  17. Alothman, M.; Bhat, R.; Karim, A.A. Antioxidant capacity and phenolic content of selected tropical fruits from malaysia, extracted with different solvents. Food Chem. 2009, 115, 785–788. [Google Scholar] [CrossRef]
  18. Farrés-Cebrián, M.; Seró, R.; Saurina, J.; Núñez, O. Hplc-uv polyphenolic profiles in the classification of olive oils and other vegetable oils via principal component analysis. Separations 2016, 3, 33. [Google Scholar] [CrossRef]
  19. Alam, M.N.; Bristi, N.J.; Rafiquzzaman, M. Review on in vivo and in vitro methods evaluation of antioxidant activity. Saudi Pharm. J. 2013, 21, 143–152. [Google Scholar] [CrossRef]
  20. Zhong, B.; Robinson, N.A.; Warner, R.D.; Barrow, C.J.; Dunshea, F.R.; Suleria, H.A. Lc-esi-qtof-ms/ms characterization of seaweed phenolics and their antioxidant potential. Mar. Drugs 2020, 18, 331. [Google Scholar] [CrossRef]
  21. Salar, R.K.; Purewal, S.S.; Sandhu, K.S. Relationships between DNA damage protection activity, total phenolic content, condensed tannin content and antioxidant potential among indian barley cultivars. Biocatal. Agric. Biotechnol. 2017, 11, 201–206. [Google Scholar] [CrossRef]
  22. Song, K.-M.; Ha, S.J.; Lee, J.-E.; Kim, S.-H.; Kim, Y.H.; Kim, Y.; Hong, S.P.; Jung, S.K.; Lee, N.H. High yield ultrasonication extraction method for Undaria pinnatifida sporophyll and its anti-inflammatory properties associated with ap-1 pathway suppression. LWT—Food Sci. Technol. 2015, 64, 1315–1322. [Google Scholar] [CrossRef]
  23. Magnusson, M.; Yuen, A.K.; Zhang, R.; Wright, J.T.; Taylor, R.B.; Maschmeyer, T.; de Nys, R. A comparative assessment of microwave assisted (mae) and conventional solid-liquid (sle) techniques for the extraction of phloroglucinol from brown seaweed. Algal Res. 2017, 23, 28–36. [Google Scholar] [CrossRef]
  24. Dang, T.T.; Bowyer, M.C.; Van Altena, I.A.; Scarlett, C. Comparison of chemical profile and antioxidant properties of the brown algae. Int. J. Food Sci. Technol. 2018, 53, 174–181. [Google Scholar] [CrossRef]
  25. Subbiah, V.; Xie, C.D.; Dunshea, F.R.; Barrow, C.J.; Suleria, H.A.R. The quest for phenolic compounds from seaweed: Nutrition, biological activities and applications. Food Rev. Int. 2022, 1–28. [Google Scholar] [CrossRef]
  26. Al-Matani, S.K.; Al-Wahaibi, R.N.S.; Hossain, M.A. Total flavonoids content and antimicrobial activity of crude extract from leaves of Ficus sycomorus native to sultanate of oman. Karbala Int. J. Mod. Sci. 2015, 1, 166–171. [Google Scholar] [CrossRef]
  27. Yusoff, I.M.; Mat Taher, Z.; Rahmat, Z.; Chua, L.S. A review of ultrasound-assisted extraction for plant bioactive compounds: Phenolics, flavonoids, thymols, saponins and proteins. Food Res. Int. 2022, 157, 111268. [Google Scholar] [CrossRef]
  28. Sobuj, M.K.A.; Islam, M.; Haque, M.; Alam, M.; Rafiquzzaman, S.M. Evaluation of bioactive chemical composition, phenolic, and antioxidant profiling of different crude extracts of Sargassum coriifolium and Hypnea pannosa seaweeds. J. Food Meas. Charact. 2021, 15, 1653–1665. [Google Scholar] [CrossRef]
  29. Fanali, C.; Tripodo, G.; Russo, M.; Della Posta, S.; Pasqualetti, V.; De Gara, L. Effect of solvent on the extraction of phenolic compounds and antioxidant capacity of hazelnut kernel. Electrophoresis 2018, 39, 1683–1691. [Google Scholar] [CrossRef]
  30. Ford, L.; Theodoridou, K.; Sheldrake, G.N.; Walsh, P.J. A critical review of analytical methods used for the chemical characterisation and quantification of phlorotannin compounds in brown seaweeds. Phytochem. Anal. 2019, 30, 587–599. [Google Scholar] [CrossRef]
  31. Fogliano, V.; Verde, V.; Randazzo, G.; Ritieni, A. Method for measuring antioxidant activity and its application to monitoring the antioxidant capacity of wines. J. Agric. Food Chem. 1999, 47, 1035–1040. [Google Scholar] [CrossRef]
  32. Lutz, M.; Hernández, J.; Henríquez, C. Phenolic content and antioxidant capacity in fresh and dry fruits and vegetables grown in chile. CyTA—J. Food 2015, 13, 541–547. [Google Scholar]
  33. Tapia-Salazar, M.; Arévalo-Rivera, I.G.; Maldonado-Muñiz, M.; Garcia-Amezquita, L.E.; Nieto-López, M.G.; Ricque-Marie, D.; Cruz-Suárez, L.E.; Welti-Chanes, J. The dietary fiber profile, total polyphenol content, functionality of silvetia compressa and ecklonia arborea, and modifications induced by high hydrostatic pressure treatments. Food Bioprocess. Technol. 2019, 12, 512–523. [Google Scholar] [CrossRef]
  34. Marinova, G.; Batchvarov, V. Evaluation of the methods for determination of the free radical scavenging activity by dpph. Bulg. J. Agric. Sci. 2011, 17, 11–24. [Google Scholar]
  35. Seo, Y.-W.; Park, K.-E.; Nam, T.-J. Isolation of a new chromene from the brown alga Sargassum thunbergii. Bull. Korean Chem. Soc. 2007, 28, 1831–1833. [Google Scholar]
  36. Arrieta, M.P.; López de Dicastillo, C.; Garrido, L.; Roa, K.; Galotto, M.J. Electrospun pva fibers loaded with antioxidant fillers extracted from Durvillaea antarctica algae and their effect on plasticized pla bionanocomposites. Eur. Polym. J. 2018, 103, 145–157. [Google Scholar] [CrossRef]
  37. Dasgupta, A.; Klein, K. Chapter 2—Methods for measuring oxidative stress in the laboratory. In Antioxidants in Food, Vitamins and Supplements; Dasgupta, A., Klein, K., Eds.; Elsevier: San Diego, CA, USA, 2014; pp. 19–40. [Google Scholar]
  38. Trkan, K.; Kasim, T.; Bircan, e.; Murat, K. DNA damage protecting activity and in vitro antioxidant potential of the methanol extract of cherry (Prunus avium L.). J. Med. Plants Res. 2014, 8, 715–726. [Google Scholar] [CrossRef]
  39. Dhingra, N.; Sharma, R.; Kar, A. Evaluation of the antioxidant activities of Prunus domestica whole fruit: An in vitro study. Int. J. Pharm. Pharm. Sci. 2014, 6, 271–276. [Google Scholar]
  40. Zakaria, N.A.; Ibrahim, D.; Sulaiman, S.F.; Supardy, A. Assessment of antioxidant activity, total phenolic content and in vitro toxicity of malaysian red seaweed, Acanthophora spicifera. J. Chem. Pharm. Res. 2011, 3, 182–191. [Google Scholar]
  41. Abirami, R.; Kowsalya, S. Quantification and correlation study on derived phenols and antioxidant activity of seaweeds from gulf of mannar. J. Herbs Spices Med. Plants 2017, 23, 9–17. [Google Scholar] [CrossRef]
  42. Matanjun, P.; Mohamed, S.; Mustapha, N.M.; Muhammad, K.; Ming, C.H. Antioxidant activities and phenolics content of eight species of seaweeds from north borneo. J. Appl. Phycol. 2008, 20, 367–373. [Google Scholar] [CrossRef]
  43. Steinberg, P.D. Biogeographical variation in brown algal polyphenolics and other secondary metabolites—Comparison between temperate australasia and north-america. Oecologia 1989, 78, 373–382. [Google Scholar] [CrossRef]
  44. Lever, J.; Brkljača, R.; Kraft, G.; Urban, S. Natural products of marine macroalgae from south eastern australia, with emphasis on the port phillip bay and heads regions of victoria. Mar. Drugs 2020, 18, 142. [Google Scholar] [CrossRef]
  45. Do, Q.D.; Angkawijaya, A.E.; Tran-Nguyen, P.L.; Huynh, L.H.; Soetaredjo, F.E.; Ismadji, S.; Ju, Y.H. Effect of extraction solvent on total phenol content, total flavonoid content, and antioxidant activity of Limnophila aromatica. J. Food Drug Anal. 2014, 22, 296–302. [Google Scholar] [CrossRef]
  46. Babbar, N.; Oberoi, H.S.; Sandhu, S.K.; Bhargav, V.K. Influence of different solvents in extraction of phenolic compounds from vegetable residues and their evaluation as natural sources of antioxidants. J. Food Sci. Technol. 2014, 51, 2568–2575. [Google Scholar] [CrossRef] [PubMed]
  47. Sultana, B.; Anwar, F.; Ashraf, M. Effect of extraction solvent/technique on the antioxidant activity of selected medicinal plant extracts. Molecules 2009, 14, 2167–2180. [Google Scholar] [CrossRef] [PubMed]
  48. Anokwuru, C.; Anyasor, G.; Ajibaye, O.; Fakoya, O.; Okebugwu, P. Effect of extraction solvents on phenolic, flavonoid and antioxidant activities of three nigerian medicinal plants. Nat. Sci. 2011, 9, 53–61. [Google Scholar]
  49. Das, P.R.; Islam, M.T.; Lee, S.-H.; Lee, M.-K.; Kim, J.-B.; Eun, J.-B. Uplc-dad-qtof/ms analysis of green tea phenolic metabolites in their free, esterified, glycosylated, and cell wall-bound forms by ultra-sonication, agitation, and conventional extraction techniques. LWT 2020, 127, 109440. [Google Scholar] [CrossRef]
  50. Rodrigues, S.; Pinto, G.A.S. Ultrasound extraction of phenolic compounds from coconut (Cocos nucifera) shell powder. J. Food Eng. 2007, 80, 869–872. [Google Scholar] [CrossRef]
  51. Harukaze, A.; Murata, M.; Homma, S. Analyses of free and bound phenolics in rice. Food Sci. Technol. Res. 1999, 5, 74–79. [Google Scholar] [CrossRef]
  52. Ramón-Gonçalves, M.; Gómez-Mejía, E.; Rosales-Conrado, N.; León-González, M.E.; Madrid, Y.J.W.M. Extraction, identification and quantification of polyphenols from spent coffee grounds by chromatographic methods and chemometric analyses. Waste Manag. 2019, 96, 15–24. [Google Scholar] [CrossRef]
  53. Escobar-Avello, D.; Lozano-Castellón, J.; Mardones, C.; Pérez, A.J.; Saéz, V.; Riquelme, S.; von Baer, D.; Vallverdú-Queralt, A.J.M. Phenolic profile of grape canes: Novel compounds identified by lc-esi-ltq-orbitrap-ms. Molecules 2019, 24, 3763. [Google Scholar]
  54. Wang, X.; Yan, K.; Ma, X.; Li, W.; Chu, Y.; Guo, J.; Li, S.; Zhou, S.; Zhu, Y.; Liu, C. Simultaneous determination and pharmacokinetic study of protocatechuic aldehyde and its major active metabolite protocatechuic acid in rat plasma by liquid chromatography-tandem mass spectrometry. J. Chromatogr. Sci. 2016, 54, 697–705. [Google Scholar] [CrossRef]
  55. Bontpart, T.; Marlin, T.; Vialet, S.; Guiraud, J.L.; Pinasseau, L.; Meudec, E.; Sommerer, N.; Cheynier, V.; Terrier, N. Two shikimate dehydrogenases, vvsdh3 and vvsdh4, are involved in gallic acid biosynthesis in grapevine. J. Exp. Bot. 2016, 67, 3537–3550. [Google Scholar] [CrossRef] [PubMed]
  56. Rajauria, G.; Foley, B.; Abu-Ghannam, N. Identification and characterization of phenolic antioxidant compounds from brown irish seaweed himanthalia elongata using lc-dad-esi-ms/ms. Innov. Food Sci. Emerg. Technol. 2016, 37, 261–268. [Google Scholar] [CrossRef]
  57. Wekre, M.E.; Kåsin, K.; Underhaug, J.; Holmelid, B.; Jordheim, M.J.A. Quantification of polyphenols in seaweeds: A case study of ulva intestinalis. Antioxidants 2019, 8, 612. [Google Scholar] [CrossRef] [PubMed]
  58. Karamac, M.; Kosiñska, A.; Pegg, R. Content of gallic acid in selected plant extracts. Pol. J. Food Nutr. Sci. 2006, 15, 55. [Google Scholar]
  59. Genwali, G.R.; Acharya, P.P.; Rajbhandari, M. Isolation of gallic acid and estimation of total phenolic content in some medicinal plants and their antioxidant activity. Nepal J. Sci. Technol. 2013, 14, 95–102. [Google Scholar] [CrossRef]
  60. Verma, S.; Singh, A.; Mishra, A. Gallic acid: Molecular rival of cancer. Environ. Toxicol. Pharmacol. 2013, 35, 473–485. [Google Scholar] [CrossRef]
  61. Iqbal, Y.; Ponnampalam, E.N.; Suleria, H.A.R.; Cottrell, J.J.; Dunshea, F.R. Lc-esi/qtof-ms profiling of chicory and lucerne polyphenols and their antioxidant activities. Antioxidants 2021, 10, 932. [Google Scholar] [CrossRef]
  62. Zanta, C.; Martínez-Huitle, C.A. Degradation of 2-hydroxybenzoic acid by advanced oxidation processes. Braz. J. Chem. Eng. 2009, 26, 503–513. [Google Scholar] [CrossRef]
  63. Suleria, H.A.; Barrow, C.J.; Dunshea, F.R. Screening and characterization of phenolic compounds and their antioxidant capacity in different fruit peels. Foods 2020, 9, 1206. [Google Scholar] [CrossRef]
  64. Wang, J.; Jia, Z.; Zhang, Z.; Wang, Y.; Liu, X.; Wang, L.; Lin, R. Analysis of chemical constituents of melastoma dodecandrum lour. By uplc-esi-q-exactive focus-ms/ms. Molecules 2017, 22, 476. [Google Scholar] [CrossRef]
  65. Mathew, S.; Abraham, T.E. Bioconversions of ferulic acid, an hydroxycinnamic acid. Crit. Rev. Microbiol. 2006, 32, 115–125. [Google Scholar] [CrossRef]
  66. Agregán, R.; Munekata, P.E.S.; Franco, D.; Dominguez, R.; Carballo, J.; Lorenzo, J.M. Phenolic compounds from three brown seaweed species using lc-dad–esi-ms/ms. Food Res. Int. 2017, 99, 979–985. [Google Scholar] [CrossRef]
  67. Rajauria, G. Optimization and validation of reverse phase hplc method for qualitative and quantitative assessment of polyphenols in seaweed. J. Pharm. Biomed. Anal. 2018, 148, 230–237. [Google Scholar] [CrossRef]
  68. Kumar, N.; Pruthi, V. Potential applications of ferulic acid from natural sources. Biotechnol. Rep. 2014, 4, 86–93. [Google Scholar] [CrossRef]
  69. Ou, S.Y.; Kwok, K.C. Ferulic acid: Pharmaceutical functions, preparation and applications in foods. J. Sci. Food Agric. 2004, 84, 1261–1269. [Google Scholar] [CrossRef]
  70. Montanari, L.; Perretti, G.; Natella, F.; Guidi, A.; Fantozzi, P. Organic and phenolic acids in beer. LWT—Food Sci. Technol. 1999, 32, 535–539. [Google Scholar] [CrossRef]
  71. Strassburg, C.P.; Kalthoff, S. Chapter 60—Coffee and gastrointestinal glucuronosyltransferases. In Coffee in Health and Disease Prevention; Preedy, V.R., Ed.; Academic Press: San Diego, CA, USA, 2015; pp. 535–543. [Google Scholar]
  72. Moselhy, S.S.; Razvi, S.S.; ALshibili, F.A.; Kuerban, A.; Hasan, M.N.; Balamash, K.S.; Huwait, E.A.; Abdulaal, W.H.; Al-Ghamdi, M.A.; Kumosani, T.A.; et al. M-coumaric acid attenuates non-catalytic protein glycosylation in the retinas of diabetic rats. J. Pestic. Sci. 2018, 43, 180–185. [Google Scholar] [CrossRef] [PubMed]
  73. Castro, C.B.; Luz, L.R.; Guedes, J.A.C.; Porto, D.D.; Silva, M.F.S.; Silva, G.S.; Riceli, P.R.V.; Canuto, K.M.; Brito, E.S.; Zampieri, D.S.; et al. Metabolomics-based discovery of biomarkers with cytotoxic potential in extracts of myracrodruon urundeuva. J. Braz. Chem. Soc. 2020, 31, 775–787. [Google Scholar] [CrossRef]
  74. Wang, Y.; Vorsa, N.; Harrington, P.B.; Chen, P. Nontargeted metabolomic study on variation of phenolics in different cranberry cultivars using uplc-im—Hrms. J. Agric. Food Chem. 2018, 66, 12206–12216. [Google Scholar] [CrossRef]
  75. Singh, P.; Arif, Y.; Bajguz, A.; Hayat, S. The role of quercetin in plants. Plant Physiol. Biochem. 2021, 166, 10–19. [Google Scholar] [CrossRef]
  76. Zheng, W.; Wang, S.Y. Oxygen radical absorbing capacity of phenolics in blueberries, cranberries, chokeberries, and lingonberries. J. Agric. Food Chem. 2003, 51, 502–509. [Google Scholar] [CrossRef] [PubMed]
  77. Olate-Gallegos, C.; Barriga, A.; Vergara, C.; Fredes, C.; Garcia, P.; Gimenez, B.; Robert, P. Identification of polyphenols from chilean brown seaweeds extracts by lc-dad-esi-ms/ms. J. Aquat. Food Prod. Technol. 2019, 28, 375–391. [Google Scholar] [CrossRef]
  78. Zhao, X.; Zhang, S.; Liu, D.; Yang, M.; Wei, J. Analysis of flavonoids in dalbergia odorifera by ultra-performance liquid chromatography with tandem mass spectrometry. Molecules 2020, 25, 389. [Google Scholar] [CrossRef] [PubMed]
  79. Gampe, N.; Darcsi, A.; Lohner, S.; Béni, S.; Kursinszki, L. Characterization and identification of isoflavonoid glycosides in the root of spiny restharrow (ononis spinosa l.) by hplc-qtof-ms, hplc–ms/ms and nmr. J. Pharm. Biomed. Anal. 2016, 123, 74–81. [Google Scholar] [CrossRef]
  80. Ham, S.A.; Hwang, J.S.; Kang, E.S.; Yoo, T.; Lim, H.H.; Lee, W.J.; Paek, K.S.; Seo, H.G. Ethanol extract of dalbergia odorifera protects skin keratinocytes against ultraviolet b-induced photoaging by suppressing production of reactive oxygen species. Biosci. Biotechnol. Biochem. 2015, 79, 760–766. [Google Scholar] [CrossRef]
  81. Liu, R.; Sun, J.; Bi, K.; Guo, D.-a. Identification and determination of major flavonoids in rat serum by hplc–uv and hplc–ms methods following oral administration of dalbergia odorifera extract. J. Chromatogr. B 2005, 829, 35–44. [Google Scholar] [CrossRef]
  82. Al-Snafi, A.E. Chemical constituents and pharmacological effects of dalbergia sissoo-a review. IOSR J. Pharm. 2017, 7, 59–71. [Google Scholar] [CrossRef]
  83. Zeng, Y.; Lu, Y.; Chen, Z.; Tan, J.; Bai, J.; Li, P.; Wang, Z.; Du, S. Rapid characterization of components in bolbostemma paniculatum by uplc/ltq-orbitrap msn analysis and multivariate statistical analysis for herb discrimination. Molecules 2018, 23, 1155. [Google Scholar] [CrossRef]
  84. Parama, D.; Girisa, S.; Khatoon, E.; Kumar, A.; Alqahtani, M.S.; Abbas, M.; Sethi, G.; Kunnumakkara, A.B. An overview of the pharmacological activities of scopoletin against different chronic diseases. Pharmacol. Res. 2022, 179, 106202. [Google Scholar] [CrossRef]
  85. Gnonlonfin, G.J.B.; Sanni, A.; Brimer, L. Review scopoletin—A coumarin phytoalexin with medicinal properties. Crit. Rev. Plant Sci. 2012, 31, 47–56. [Google Scholar] [CrossRef]
  86. Jamuna, S.; Karthika, K.; Paulsamy, S.; Thenmozhi, K.; Kathiravan, S.; Venkatesh, R. Confertin and scopoletin from leaf and root extracts of hypochaeris radicata have anti-inflammatory and antioxidant activities. Ind. Crops Prod. 2015, 70, 221–230. [Google Scholar] [CrossRef]
  87. Stella, L.; De Rosso, M.; Panighel, A.; Vedova, A.D.; Flamini, R.; Traldi, P. Collisionally induced fragmentation of [M–H]− species of resveratrol and piceatannol investigated by deuterium labelling and accurate mass measurements. Rapid Commun. Mass Spectrom. 2008, 22, 3867–3872. [Google Scholar] [CrossRef] [PubMed]
  88. Frémont, L. Biological effects of resveratrol. Life Sci. 2000, 66, 663–673. [Google Scholar] [CrossRef] [PubMed]
  89. Karthik, R.; Manigandan, V.; Sheeba, R.; Saravanan, R.; Rajesh, P.R. Structural characterization and comparative biomedical properties of phloroglucinol from indian brown seaweeds. J. Appl. Phycol. 2016, 28, 3561–3573. [Google Scholar] [CrossRef]
  90. Rodríguez-bernaldo De Quirós, A.; Frecha-ferreiro, S.; Vidal-pérez, A.M.; López-hernández, J. Antioxidant compounds in edible brown seaweeds. Eur. Food Res. Technol. 2010, 231, 495–498. [Google Scholar] [CrossRef]
  91. Dewick, P.M.; Haslam, E. Phenol biosynthesis in higher plants. Gallic acid. Biochem. J. 1969, 113, 537–542. [Google Scholar] [CrossRef]
  92. Khan, M.K.; Abert-Vian, M.; Fabiano-Tixier, A.-S.; Dangles, O.; Chemat, F. Ultrasound-assisted extraction of polyphenols (flavanone glycosides) from orange (Citrus sinensis L.) peel. Food Chem. 2010, 119, 851–858. [Google Scholar] [CrossRef]
  93. Metsämuuronen, S.; Sirén, H. Bioactive phenolic compounds, metabolism and properties: A review on valuable chemical compounds in scots pine and norway spruce. Phytochem. Rev. 2019, 18, 623–664. [Google Scholar] [CrossRef]
  94. Badmus, U.O.; Taggart, M.A.; Boyd, K.G. The effect of different drying methods on certain nutritionally important chemical constituents in edible brown seaweeds. J. Appl. Phycol. 2019, 31, 3883–3897. [Google Scholar] [CrossRef]
  95. Čagalj, M.; Skroza, D.; Tabanelli, G.; Özogul, F.; Šimat, V. Maximizing the antioxidant capacity of padina pavonica by choosing the right drying and extraction methods. Processes 2021, 9, 587. [Google Scholar] [CrossRef]
  96. Gulsunoglu, Z.; Karbancioglu-Guler, F.; Raes, K.; Kilic-Akyilmaz, M. Soluble and insoluble-bound phenolics and antioxidant activity of various industrial plant wastes. Int. J. Food Prop. 2019, 22, 1501–1510. [Google Scholar] [CrossRef]
  97. Subbiah, V.; Zhong, B.; Nawaz, M.A.; Barrow, C.J.; Dunshea, F.R.; Suleria, H.A.R. Screening of phenolic compounds in australian grown berries by lc-esi-qtof-ms/ms and determination of their antioxidant potential. Antioxidants 2020, 10, 26. [Google Scholar] [CrossRef] [PubMed]
  98. Mussatto, S.I.; Ballesteros, L.F.; Martins, S.; Teixeira, J.A. Extraction of antioxidant phenolic compounds from spent coffee grounds. Sep. Purif. Technol. 2011, 83, 173–179. [Google Scholar] [CrossRef]
  99. Ali, A.; Wu, H.; Ponnampalam, E.N.; Cottrell, J.J.; Dunshea, F.R.; Suleria, H.A.R. Comprehensive profiling of most widely used spices for their phenolic compounds through lc-esi-qtof-ms(2) and their antioxidant potential. Antioxidants 2021, 10, 721. [Google Scholar] [CrossRef] [PubMed]
  100. Vissers, A.M.; Caligiani, A.; Sforza, S.; Vincken, J.P.; Gruppen, H. Phlorotannin composition of laminaria digitata. Phytochem. Anal. 2017, 28, 487–495. [Google Scholar] [CrossRef] [PubMed]
  101. Stern, J.L.; Hagerman, A.E.; Steinberg, P.D.; Winter, F.C.; Estes, J.A. A new assay for quantifying brown algal phlorotannins and comparisons to previous methods. J. Chem. Ecol. 1996, 22, 1273–1293. [Google Scholar] [CrossRef]
  102. Margraf, T.; Karnopp, A.R.; Rosso, N.D.; Granato, D. Comparison between folin-ciocalteu and prussian blue assays to estimate the total phenolic content of juices and teas using 96-well microplates. J. Food Sci. 2015, 80, C2397–C2403. [Google Scholar] [CrossRef] [PubMed]
  103. Nebesny, E.; Budryn, G. Antioxidative activity of green and roasted coffee beans as influenced by convection and microwave roasting methods and content of certain compounds. Eur. Food Res. Technol. 2003, 217, 157–163. [Google Scholar] [CrossRef]
  104. Benzie, I.F.; Strain, J.J. The ferric reducing ability of plasma (frap) as a measure of “antioxidant power”: The frap assay. Anal. Biochem. 1996, 239, 70–76. [Google Scholar] [CrossRef]
  105. Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Antioxidant activity applying an improved abts radical cation decolorization assay. Free Radic. Biol. Med. 1999, 26, 1231–1237. [Google Scholar] [CrossRef]
  106. Smirnoff, N.; Cumbes, Q.J. Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry 1989, 28, 1057–1060. [Google Scholar] [CrossRef]
  107. Dinis, T.C.; Maderia, V.M.; Almeida, L.M. Action of phenolic derivatives (acetaminophen, salicylate, and 5-aminosalicylate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Arch. Biochem. Biophys. 1994, 315, 161–169. [Google Scholar] [CrossRef] [PubMed]
  108. Ferreira, I.C.F.R.; Baptista, P.; Vilas-Boas, M.; Barros, L. Free-radical scavenging capacity and reducing power of wild edible mushrooms from northeast portugal: Individual cap and stipe activity. Food Chem. 2007, 100, 1511–1516. [Google Scholar] [CrossRef]
  109. Prieto, P.; Pineda, M.; Aguilar, M. Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: Specific application to the determination of vitamin e. Anal. Biochem. 1999, 269, 337–341. [Google Scholar] [CrossRef] [PubMed]
  110. Allwood, J.W.; Evans, H.; Austin, C.; McDougall, G.J. Extraction, enrichment, and lc-msn-based characterization of phlorotannins and related phenolics from the brown seaweed, ascophyllum nodosum. Mar. Drugs 2020, 18, 448. [Google Scholar] [CrossRef] [PubMed]
  111. Zhu, Z.; Zhong, B.; Yang, Z.; Zhao, W.; Shi, L.; Aziz, A.; Rauf, A.; Aljohani, A.S.M.; Alhumaydhi, F.A.; Suleria, H.A.R. Lc-esi-qtof-ms/ms characterization and estimation of the antioxidant potential of phenolic compounds from different parts of the lotus (nelumbo nucifera) seed and rhizome. ACS Omega 2022, 7, 14630–14642. [Google Scholar] [CrossRef] [PubMed]
  112. Gu, C.; Suleria, H.A.R.; Dunshea, F.R.; Howell, K. Dietary lipids influence bioaccessibility of polyphenols from black carrots and affect microbial diversity under simulated gastrointestinal digestion. Antioxidants 2020, 9, 762. [Google Scholar] [CrossRef] [PubMed]
Pharmaceuticals 16 00773 g001 550
Figure 1. 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.
Figure 1. 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.
Pharmaceuticals 16 00773 g001
Pharmaceuticals 16 00773 g002 550
Figure 2. 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.
Figure 2. 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.
Pharmaceuticals 16 00773 g002
Pharmaceuticals 16 00773 g003 550
Figure 3. 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.
Figure 3. 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.
Pharmaceuticals 16 00773 g003
Pharmaceuticals 16 00773 g004 550
Figure 4. 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.
Figure 4. 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.
Pharmaceuticals 16 00773 g004
Pharmaceuticals 16 00773 g005 550
Figure 5. 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.
Figure 5. 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.
Pharmaceuticals 16 00773 g005
Pharmaceuticals 16 00773 g006 550
Figure 6. (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.
Figure 6. (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.
Pharmaceuticals 16 00773 g006
Pharmaceuticals 16 00773 g007 550
Figure 7. 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.
Figure 7. 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.
Pharmaceuticals 16 00773 g007
Table
Table 1. Estimation of freeze-dried total phenolics of seaweed species extracted by the conventional and non-conventional method of ultrasonication.
Table 1. Estimation of freeze-dried total phenolics of seaweed species extracted by the conventional and non-conventional method of ultrasonication.
SamplesSolventsTPC
(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% ACE19.35 ± 0.09 Ba2.28 ± 0.02 HIb0.67 ± 0.06 EFc1.96 ± 0.08 Fb2.61 ± 0.30 Je11.21 ± 0.7 Ba
70% MeOH22.74 ± 0.28 Ba3.33 ± 0.02 E–Gb-1.02 ± 0.04 Ia8.61 ± 0.08 Bb3.86 ± 0.1 Hc
70% EtOH28.92 ± 1.33 Aa3.82 ± 0.07 DEa2.21 ± 0.01 Ba1.63 ± 0.02 Ga8.11 ± 0.61 BCa14.2 ± 0.63 Aa
EA1.66 ± 0.09 Ob7.13 ± 0.9 Cb0.94 ± 0.07 DEa4.15 ± 0.01 Dc5.05 ± 0.25 Ha-
Phyllospora comosa70% ACE16.11 ± 0.85 Fb1.86 ± 0.01 IJc0.94 ± 0.07 DEb4.96 ± 0.44 Ca6.18 ± 0.29 DEFb8.57 ± 0.89 Cb
70% MeOH16.85 ± 0.12 Db3.43 ± 0.06 D–Fb2.86 ± 0.12 Aa0.67 ± 0.01 JKb12.62 ± 0.26 Aa6.58 ± 0.17 Ea
70% EtOH12.41 ± 0.03 Hc3.62 ± 0.03 D-Fb2.18 ± 0.06 Ba0.46± 0.01 LMd3.2 ± 0.22 Ic6.36 ± 0.41 Fb
EA2.23 ± 0.04 Pd3.54 ± 0.10 D–Fc0.77 ± 0.06 Eb6.65 ± 0.67 Bb0.13 ± 0.6 Lc-
Sargassum sp.70% ACE14.6 ± 0.72 Gc0.67 ± 0.04 Le0.27 ± 0.02 Gd0.89 ± 0.05 Id6.5 ± 0.07 DEFa7.64 ± 0.25 Dc
70% MeOH16.36 ± 0.34 Ec1.39 ± 0.01 JKc-0.68 ± 0.04 JKb7.77 ± 0.16 Cc5.24 ± 0.37 Gb
70% EtOH17.52 ± 0.57 Cb2.79 ± 0.02 GHd0.64 ± 0.03 EFc0.72 ± 0.11 Jc6.85 ± 0.19 Db3.39 ± 0.25 Jd
EA0.34 ± 0.03 Qe2.6 ± 0.04 Hd-3.24 ± 0.34 Ed1.18 ± 0.3 Kb-
Ecklonia radiata70% ACE11.19 ± 0.41 Id0.85 ± 0.01 KLd1.29 ± 0.01 CDa1.02 ± 0.1 Ic5.86 ± 0.25 FGc2.5 ± 0.04 Le
70% MeOH12.19 ± 0.41 Id11.15 ± 0.12 Ba0.92 ± 0.09 DEc0.96 ± 0.07 a8 ± 0.37 BCc3.5 ± 0.1 IJd
70% EtOH9.03 ± 0.11 Kd1.65 ± 0.04 Je-1.18 ± 0.02 Hb6.13 ± 0.14 EFb0.71 ± 0.01 Me
EA1.54 ± 0.01 Oc4.04 ± 0.01 Dc-8.29 ± 0.47 Aa0.96 ± 0.13 Kb-
Durvillaea sp.70% ACE9.98 ± 0.68 Je3.35 ± 0.03 EFa1.04 ± 0.01 Db0.57 ± 0.02 KLe5.36 ± 0.14 GHd6.72 ± 0.26 Ed
70% MeOH4.45 ± 0.09 Ne1.32 ± 0.03 JKc2.12 ± 0.07 Bb0.35 ± 0.02 Mc3.87 ± 0.47 Id2.82 ± 0.25 Ke
70% EtOH8.6 ± 0.45 Le3.15 ± 0.01 FGc1.9 ± 0.05 Cb0.4 ± 0.01 Md6.63 ± 0.58 DEb3.63 ± 0.3 Ic
EA7.26 ± 0.03 Ma18.23 ± 0.17 Aa-8.22 ± 0.18 Aa0.48 ± 0.04 KLb-
Conventional Extraction
Cystophora sp.70% ACE18.27 ± 1.06 Ba2.46 ± 0.03 Da0.15 ± 0.01 FGc0.57 ± 0.03 HIa0.48 ± 0.21 Ke7.17 ± 0.55 Db
70% MeOH18.59 ± 0.54 Aa1.83 ± 0.01 Ea2.55 ± 0.07 Ba0.94 ± 0.26 Fb7.35 ± 0.1 Aa8.94 ± 0.42 Ba
70% EtOH15.49 ± 0.59 Ca2.48 ± 0.04 Da1.40 ± 0.07 Da0.79 ± 0.14 Gb6.2 ± 0.25 Cb8.44 ± 0.55 Ca
EA0.96 ± 0.05 La2.51 ± 0.15 Cc0.17 ± 0.02 FGb---
Sargassum sp.70% ACE13.93 ± 0.45 D0.34 ± 0.01 Ke0.28 ± 0.03 EFb0.65 ± 0.01 Ha4.74 ± 0.15 Ea3.05 ± 0.13 Gd
70% MeOH10.37 ± 0.63 Fc1.38 ± 0.02 Kb0.43 ± 0.11 Ec0.54 ± 0.03 HIc7.03 ± 0.08 Bb2.91 ± 0.19 Hb
70% EtOH8.89 ± 0.19 Gc0.96 ± 0.06 Kd0.13 ± 0.01 FGc0.58 ± 0.01 HIc3.7 ± 0.06 Fd0.69 ± 0.2 Kd
EA0.53 ± 0.03 Mc6.35 ± 0.01 Bb0.02 ± 0.01 Gc6.32 ± 0.8 Bb0.12 ± 0.06 Lc-
Phyllospora comosa70% ACE13.72 ± 0.50 Db1.07 ± 0.02 Fb0.59 ± 0.01 Hd0.38 ± 0.01 JKb2.26 ± 0.35 Id1.87 ± 0.12 Je
70% MeOH11.03 ± 0.81 Eb0.22 ± 0.01 Lc0.14 ± 0.05 EFd0.33 ± 0.01 Kd1.48 ± 0.22 Jd1.92 ± 0.01 Jd
70% EtOH10.28 ± 0.59 Fb0.91 ± 0.02 Gb-0.47 ± 0.03 IJd6.95 ± 0.09 Ba4.69 ± 0.2 Eb
EA0.68 ± 0.01 Mc6.52 ± 0.03 Aa-6.01 ± 0.01 Cc2.22 ± 0.2 Ia-
Ecklonia radiata70% ACE10.78 ± 0.31 Gc0.35 ± 0.03 Jd-0.59 ± 0.03 HIa2.6 ± 0.05 Hc3.6 ± 0.3 Fc
70% MeOH10.63 ± 0.38 Hd0.12 ± 0.04 Lc-1.12 ± 0.12 Ea1.46 ± 0.1 Jd2.5 ± 0.04 Ic
70% EtOH8.74 ± 0.21 Id0.34 ± 0.05 Gb-0.91 ± 0.07 FGa4.94 ± 0.7 Dc2.82 ± 0.02 Hc
EA-0.11 ± 0.02 Aa27.54 ± 0.23 Aa6.57 ± 0.3 Aa0.46 ± 0.3 Kb-
Durvillaea sp.70% ACE8.13 ± 0.20 Hd0.99 ± 0.03 Ic0.8 ± 0.22 Da0.32 ± 0.07 Kb2.8 ± 0.09 Gb20.04 ± 0.23 Aa
70% MeOH4.32 ± 0.15 Ke0.04 ± 0.01 Md1.61 ± 0.09 Cb0.29 ± 0.07 Kd6.19 ± 0.01 Cc0.19 ± 0.01 Le
70% EtOH5.01 ± 0.33 Je0.78 ± 0.01 Hc0.53 ± 0.07 Eb0.39 ± 0.05 JKe4.8 ± 0.07D Ec2.91 ± 0.26 Hc
EA0.09 ± 0.01 Nd6.35 ± 0.01 Bc-0.9 ± 0.01 FGe--
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).
Table
Table 2. Estimation of freeze-dried antioxidant potential of seaweed species extracted by the conventional and non-conventional method of ultrasonication.
Table 2. Estimation of freeze-dried antioxidant potential of seaweed species extracted by the conventional and non-conventional method of ultrasonication.
SamplesSolventsDPPH
(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% ACE50.33 ± 0.07 Aa45.62 ± 0.13 Aa64.15 ± 0.20 Aa1.76 ± 0.01 BCd13.26 ± 0.22 Gd76.61 ± 0.30 Aa17.21 ± 0.03 Gc
70% MeOH43.6 ± 0.10 Ba26.08 ± 0.22 Ba58.65 ± 0.18 Ba0.96 ± 0.01 Fd62.67 ± 0.44 Bb29.23 ± 1.04 Ic16.11 ± 0.08 Hc
70% EtOH7.26 ± 0.03 Hb25.00 ± 0.22 Ca44.24 ± 0.18 Db1.04 ± 0.01 Fa-33.30 ± 1.20 Gb29.27 ± 0.14 Aa
EA1.42 ± 0.8 Nb0.35 ± 0.01 Nc6.58 ± 0.06 Pb1.45 ± 0.9 BCb9.06 ± 0.55 I20.24 ± 0.6 Kc1.95± 0.35 Pb
Phyllospora comosa70% ACE37.52 ± 0.07 Dc17.01 ± 0.12 Gc22.99 ± 0.85 Kb4.14 ± 0.11 Aa53.38 ± 1.10 Cb48.71 ± 0.76 Cc25.76 ± 0.09 Cb
70% MeOH4.76 ± 0.03 Le7.80 ± 0.23 Ic33.22 ± 0.33 Fc1.91 ± 0.02 Ba65.42 ± 0.96 Ba33.52 ± 0.60 Ga20.46 ± 0.18 Da
70% EtOH4.69 ± 0.01 Kd11.02 ± 0.19 Hd32.21 ± 0.11 Ge1.59 ± 0.01 B–Db15.38 ± 0.17 Fa40.94 ± 0.92 Da7.24 ± 0.04 Me
EA2.1 ± 0.03 Ma0.53 ± 0.04 Nd9.03 ± 0.03 Oa1.02 ± 0.04 Fb33.23 ± 0.15 H-2.90 ± 0.04 Oa
Sargassum sp. 70% ACE38.79 ± 0.06 Cb36.73 ± 0.27 Db45.16 ± 0.40 Cd1.45 ± 0.01 C–Ed16.73 ± 0.36 Ec56.01 ± 0.35 Bb28.47 ± 0.14 Ba
70% MeOH7.89 ± 0.06 Ga35.60 ± 0.21 Eb30.13 ± 0.07 Hd1.11 ± 0.01 Fc3.66 ± 0.01 Fd32.58 ± 0.63 GHb15.53 ± 0.04 Id
70% EtOH7.84 ± 0.03 Ga27.63 ± 0.16 Fb30.22 ± 0.75 Ha1.01 ± 0.01 Ga1.33 ± 0.05 Kd31.63 ± 0.46 Hc18.52 ± 0.19 Fb
EA0.62 ± 0.01 Oc0.09 ± 0.01 Mb4.86 ± 0.05 Qd0.52 ± 0.02 Hc-40.30 ± 1.26 Ea-
Ecklonia radiata70% ACE34.09 ± 0.05 Ed24.10 ± 0.12 Db24.92 ± 0.45 Jd1.87 ± 0.01 Bb134.49 ± 5.25 Aa37.82 ± 1.5 Fd11.90 ± 0.04 Jd
70% MeOH5.01 ± 0.01 Ld20.80 ± 0.09 Eb40.91 ± 0.99 Eb0.53 ± 0.01 He11.94 ± 0.01 Cb15.35 ± 0.76 Ld19.10 ± 0.08 Eb
70% EtOH5.51 ± 0.02 Ic19.20 ± 0.16 Fb21.09 ± 0.69 Lc1.87 ± 0.01 D–Fa3.23 ± 0.06 Jc26.43 ± 1.5 Jd8.61 ± 0.08 Ld
EA-0.84 ± 0.01 Mb6.75 ± 0.10 PQc0.66 ± 0.01 Ba-33.79 ± 0.42 Hb1.88 ± 0.02 Qc
Durvillaea sp. 70% ACE22.49 ± 0.04 Fe3.58 ± 0.08 Ld28.42 ± 0.07 Ie1.06 ± 0.01 Fe13.12 ± 0.16 Gd18.50 ± 0.08 Ke4.68 ± 0.16 Ne
70% MeOH5.24 ± 0.01 Jc6.39 ± 0.12 Jd17.67 ± 0.09 Ne1.16 ± 0.01 EFb18.22 ± 0.02 Dc7.01 ± 0.15 Ne11.99 ± 0.04 Je
70% EtOH5.47 ± 0.01 Ic11.39 ± 0.08 Hc18.36 ± 0.40 Mc1.01 ± 0.01 Fa14.27 ± 0.09 Fb9.36 ± 0.15 Me11.65 ± 0.06 Kc
EA2.01 ± 0.01 Ma0.25 ± 0.01 Ka6.40 ± 0.03 Qd1.92 ± 0.01 Ba-5.86 ± 0.05 Od2.00 ± 0.03 Pb
Conventional Extraction
Cystophora sp.70% ACE46.73 ± 0.12 Aa32.54 ± 0.29 Ca42.50 ± 0.22 Aa0.24 ± 0.010.70 ± 0.73 Jc77.63 ± 0.17 Aa7.92 ± 0.03 Fd
70% MeOH42.95 ± 0.12 Ca32.88 ± 0.12 Ba41.10 ± 0.15 Ba0.10 ± 0.01 Nc2.20 ± 0.74 Hc46.00 ± 0.54 Ca15.20 ± 0.14 Ba
70% EtOH46.49 ± 0.08 Ba33.48 ± 0.23 Aa42.34 ± 0.32 Aa0.37 ± 0.01 Ke3.72 ± 0.05 Gc73.79 ± 0.30 Ba22.01 ± 0.10 Aa
EA4.40 ± 0.06 Jb0.25 ± 0.01 Opb3.41 ± 0.04 Lb0.93 ± 0.01 Ee-17.59 ± 0.35 Ha2.27 ± 0.09 La
Sargassum sp.70% ACE32.94 ± 0.17 Ec30.35 ± 0.17 Db23.43 ± 0.27 Cb0.36 ± 0.02 Kc23.37 ± 0.74 Ca34.75 ± 0.52 Cb10.55 ± 0.08 Da
70% MeOH5.97 ± 0.08 Hc17.83 ± 0.23 Fb23.34 ± 0.48 Cb0.46 ± 0.01 Jc3.60 ± 0.29 Gd8.85 ± 0.17 Dd7.84 ± 0.05 Hd
70% EtOH5.87 ± 0.07 Hc16.96 ± 0.32 Gb21.53 ± 0.48 Eb0.48 ± 0.03 Jc0.39 ± 0.35 Jd14.77 ± 0.18 Ld8.91 ± 0.08 Fc
EA3.44 ± 0.19 Kc0.40 ± 0.01 Oa2.88 ± 0.03 Mc0.35 ± 0.02 Kd-16.64 ± 1.30 Ib0.43 ± 0.01 Mb
Phyllospora comosa70% ACE35.09 ± 0.09 Db12.69 ± 0.23 Hd20.24 ± 0.87 Fd0.98 ± 0.01 Da5.80 ± 0.01 Eb18.01 ± 0.63 Jd8.27 ± 0.04 Gc
70% MeOH4.99 ± 0.01 Id6.56 ± 0.12 Ld12.56 ± 0.10 Ijd0.78 ± 0.01 Fa26.63 ± 0.12 Aa13.71 ± 0.31 Ic9.94 ± 0.05 Eb
70% EtOH5.12 ± 0.01 Hd9.18 ± 0.29 Kd12.66 ± 0.07 Ijd1.19 ± 0.01 Ba4.51 ± 0.13 Fb21.69 ± 0.09 Fa11.90 ± 0.12 Cb
EA0.69 ± 0.01 Me0.17 ± 0.01 Opb2.85 ± 0.05 Ka1.02 ± 0.01 Cb--0.44 ± 0.02 Mb
Ecklonia radiata70% ACE41.96 ± 0.07 Fd20.88 ± 0.12 Ec25.35 ± 0.62 Dc0.66 ± 0.01 Ib2.17 ± 0.05 H27.91 ± 0.17 Fc8.71 ± 0.07 Ge
70% MeOH31.59 ± 0.08 Fb12.32 ± 0.12 Ic19.62 ± 0.40 Hc0.37± 0.01 Ld6.75 ± 0.15 Dc28.09 ± 0.15 Eb10.17 ± 0.07 Fc
70% EtOH29.41 ± 0.12 Gb14.06 ± 0.19 Hc13.75 ± 0.23 Ijc0.66 ± 0.01 Jd1.11 ± 0.65 Ie28.24 ± 0.23 Gc11.43 ± 0.04 Fc
EA2.51 ± 0.04 Ld0.02 ± 0.01 Pc-4.56 ± 0.07 Aa--0.14 ± 0.01 Nd
Durvillaea sp.70% ACE30.54 ± 0.06 Fe10.25 ± 0.08 Je12.86 ± 0.07 Ie0.27 ± 0.01 Md4.56 ± 0.14 Ec13.31 ± 0.09 Ke9.84 ± 0.04 Ec
70% MeOH4.73 ± 0.02 Je6.20 ± 0.20 Me12.60 ± 0.07 Ijd0.56 ± 0.02 Hb16.80 ± 0.15 Bb4.09 ± 0.04 Ne5.12 ± 0.04 Je
70% EtOH4.77 ± 0.02 Je5.83 ± 0.16 Ne12.29 ± 0.04 Je0.66 ± 0.01 Gb6.50 ± 0.23 Da5.34 ± 0.14 Me4.51 ± 0.03 Kd
EA4.82 ± 0.03 Ja0.03 ± 0.01 Pc1.87 ± 0.04 Ne0.93 ± 0.01 Ec-8.01 ± 0.53 Lc-
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).
Table
Table 3. Pearson’s correlation coefficients (r) of phenolic contents and the antioxidant capacity for ultrasonication and conventional methodologies.
Table 3. Pearson’s correlation coefficients (r) of phenolic contents and the antioxidant capacity for ultrasonication and conventional methodologies.
VariablesTPCTFCTCTDMBAPBAFDADPPHFRAPABTSFICAOH-RSATAC
Ultrasonication
TFC−0.178
TCT0.222−0.155
DMBA−0.4690.491 b−0.414
PBA0.649 a−0.2380.394−0.663
FDA0.818 a−0.2970.422−0.4210.432
DPPH0.505 b−0.349−0.170−0.2360.1780.498 b
FRAP0.731 a−0.365−0.145−0.5030.4030.600 a 0.657 a
ABTS0.837 a−0.2450.110−0.5710.510 b0.734 a0.690 a0.815 a
FICA0.202−0.0670.1180.1500.1140.2790.4060.1110.016
OH-RSA0.165−0.2700.254−0.1640.3110.0240.474 b0.1140.1610.375
TAC0.448 b−0.442−0.111−0.1930.1100.571 a0.646 a0.650 a0.562 a0.3260.120
RPA0.819 a−0.2940.286−0.4580.682 a0.771 a0.480 b0.712 a0.699 a0.3130.1820.449 b
Conventional
TFC−0.434
TCT−0.292−0.205
DMBA−0.6680.511 b0.488
PBA0.457 b−0.384−0.196−0.513
FDA0.478 b−0.195−0.133−0.3930.262
DPPH0.799 a−0.262−0.186−0.4450.1670.561 b
FRAP0.907 a−0.304−0.217−0.5380.478 a0.447 b0.824 a
ABTS0.921 b−0.280−0.257−0.5670.457 a0.451 b0.809 a0.958 a
FICA−0.460−0.1220.926 a0.537 b−0.298−0.321−0.376−0.443−0.480
OH-RSA0.247−0.444−0.153−0.3900.190−0.40−0.0210.1120.047−0.168
TAC0.761 a0.055 b−0.220−0.3170.1540.4030.809 a0.837 a0.851 a−0.393−0.063
RPA0.830 a−0.449−0.273−0.6250.628 a0.541 b0.688 a0.785 a0.787 a−0.4230.2190.685 a
a Significant correlation with p ≤ 0.01; b Significant correlation with p ≤ 0.05.
Table
Table 4. Characterization of phenolic compounds in ultrasound extracted seaweed samples by LC-ESI-QTOF-MS/MS.
Table 4. Characterization of phenolic compounds in ultrasound extracted seaweed samples by LC-ESI-QTOF-MS/MS.
No.Proposed CompoundsMolecular FormulaRT (min)Ionization (ESI+/ESI−)Molecular WeightTheoretical (m/z)Observed (m/z)Error
(ppm)		MS2 Product IonsSeaweed Samples
Phenolic acid
Hydroxybenzoic acids
14-HydroxybenzaldehydeC7H6O27.188[M – H]−122.0376121.0303121.03030.277Dabu
2Protocatechuic acid 4-O-glucosideC13H16O914.994[M – H]−316.0786315.0713315.0702−3.5153* Embu, Dmbu, Eeu
32,3-Dihydroxybenzoic acidC7H6O425.802[M – H]−154.0262153.0189153.01900.7109Eethbu
4Gallic acid 4-O-glucosideC13H16O1030.462** [M – H]−332.0768331.0695331.0691−1.2169, 125* Amu, Dmu, Aeu, Aau, Cabu, Cebu, Dmbu
5Gallic acidC7H6O531.309[M – H]−170.0225169.0152169.01541.2125* Aabu, Aebu, Ambu, Babu, Bebu, Bethbu, Bmbu, Dethbu, Eabu, Eethbu, Eeu, Eau
62-Hydroxybenzoic acidC7H6O332.019[M – H]−138.0310137.0237137.02380.793* Dabu, Dmbu
Hydroxycinnamic acids
7Feruloyl tartaric acidC14H14O94.933[M – H]−326.0652325.0579325.0575−1.2193, 149* Eau, Cau
8m-Coumaric acidC9H8O35.228[M – H]−164.0487163.0414163.0412−1.2119* Dabu, Cmbu, Debu, Dmbu, Eabu
9Caffeoyl tartaric acidC13H12O95.426[M – H]−312.0504311.0431311.04382.3161* Emu, Dmu, Amu
10Ferulic acidC10H10O45.539[M – H]−194.0585193.0512193.05162.1178, 149, 134Eau
11Isoferulic acid 3-sulfateC10H10O7S5.608[M – H]−274.0129273.0056273.0054−0.7193, 178* Aabu, Aethbu, Ambu, Bmbu, Cabu, Dabu, Amu
12Caffeic acid 3-O-glucuronideC15H16O106.388** [M – H]−356.0743355.0670355.06730.8179* Cebu, Ambu, Embu, Cethbu, Emu, Amu
13Hydroxycaffeic acidC9H8O57.205[M – H]−196.0368195.0295195.02992.1151Cebu
14Cinnamic acidC9H8O27.246[M – H]−148.0537147.0464147.04650.7103* Cebu, Aethbu, Bmbu, Cabu, Cmbu, Dabu, Eabu
15Ferulic acid 4-O-glucosideC16H20O913.750[M – H]−356.1106355.1033355.1025−2.3193, 178, 149, 134* Aabu, Babu
16Chlorogenic acidC16H18O913.985[M – H]−354.0929353.0856353.0855−0.3253, 190, 144* Dabu, Babu, Cabu, Cebu, Eabu, Eau, Aeu
171-Sinapoyl−2-feruloylgentiobioseC33H40O1814.898** [M – H]−724.2205723.2132723.21360.6529, 499* Bmbu, Cabu, Cethbu, Cmbu, Dmbu, Eebu, Aeu
18Sinapic acidC11H12O516.158** [M – H]−224.0666223.0593223.05950.9205, 163* Aabu, Cethbu, Dabu, Debu, Dmbu
19p-Coumaroyl tartaric acidC13H12O816.214[M – H]−296.0555295.0482295.04861.4115* Debu, Dmbu, Embu
20p-Coumaroyl malic acidC13H12O716.596** [M – H]−280.0596279.0523279.0512−3.9163, 119* Dmbu, Cabu, Cebu, Cethbu, Dabu, Dmbu, Emu, Aeu
211,5-Dicaffeoylquinic acidC25H24O1217.017[M – H]−516.1233515.1160515.11722.3353, 335, 191, 179* Dabu, Eabu, Eau
22Ferulic acid 4-O-glucuronideC16H18O1017.199** [M – H]−370.0895369.0822369.08363.8193* Eau, Bmu, Dabu, Debu, Embu
23p-Coumaric acid 4-O-glucosideC15H18O817.367[M – H]−326.0970325.0897325.0891−1.8163* Eabu, Dabu
24Rosmarinic acidC18H16O818.249[M – H]−360.0850359.0777359.0775−0.6179* Dmbu, Emu
25Caffeoyl glucoseC15H18O918.549[M – H]−342.0951341.0878341.08903.5179, 161* Dmbu, Dabu, Eabu
263-p-Coumaroylquinic acidC16H18O819.117[M – H]−338.0998337.0925337.09260.3265, 173, 162* Dabu, Cabu, Eabu, Eebu, Eau
27p-Coumaric acid 4-O-glucosideC15H18O721.240[M – H]−310.1024309.0951309.09510.1163Debu
285-5′-Dehydrodiferulic acidC20H18O821.657** [M – H]+386.0985385.0912385.09130.3369* Eabu, Embu, Cebu
293-Sinapoylquinic acidC18H22O1023.306[M – H]−398.1177397.1104397.11091.3233, 179Eeu
301,2,2′-TriferuloylgentiobioseC42H46O2031.165[M – H]−870.2584869.2511869.2481−3.5693, 517* Deu, Bau
Hydroxyphenyl acetic acids
313,4-Dihydroxyphenylacetic acidC8H8O46.661[M – H]−168.0432167.0359167.0350−5.4149, 123* Deu
322-Hydroxy-2-phenylacetic acidC8H8O37.182[M – H]−152.0475151.0402151.04030.7136, 92* Cebu, Aeu, Aau
Hydroxyphenylpropanoic acids
33Dihydroferulic acid 4-O-glucuronideC16H20O103.119** [M – H]−372.1090371.1017371.10190.5195* Babu, Bebu, Bmbu, Dabu, Eabu, Embu, Cethbu
34Dihydrocaffeic acid 3-O-glucuronideC15H18O107.535[M – H]−358.0913357.0840357.0837−0.8181* Bebu, Bmbu, Debu, Eabu, Eebu, Embu, Aabu
35Dihydroferulic acid 4-sulfateC10H12O7S16.763[M – H]−276.0290275.0217275.0212−1.8195, 151, 177Eebu
Flavonoids
Flavanols
36TheaflavinC29H24O123.082[M – H]−564.1254563.1181563.11983.0545* Bebu, Dabu, Eeu
37(+)-Gallocatechin 3-O-gallateC22H18O115.144[M – H]−458.0818457.0745457.0744−0.2305, 169* Cebu, Debu
38Procyanidin dimer B1C30H26O1217.032[M – H]−578.1385577.1312577.1292−3.5451Babu
393′-O-MethylcatechinC16H16O617.951** [M – H]−304.0959303.0886303.08942.6271, 163Babu
40(+)-Catechin 3-O-gallateC22H18O1020.261[M – H]−442.0879441.0806441.08111.1289, 169, 125* Embu, Eeu
41(+)-CatechinC15H14O620.437** [M – H]−290.0786289.0713289.07161.0245, 205, 179* Eeu, Dabu
42Theaflavin 3,3′-O-digallateC43H32O2024.533[M – H]−868.1448867.1375867.1373−0.2715, 563, 545* Bmu, Beu, Bau
43(−)-EpigallocatechinC15H14O730.714** [M – H]−306.0737305.0664305.06702.0261, 219* Aau, Dau, Deu, Debu, Eabu, Embu
Flavones
44Apigenin 6-C-glucosideC21H20O1016.981[M – H]−432.1077431.1004431.10152.6413, 341, 311* Dabu, Dmbu
45IsorhamnetinC16H12O719.215[M – H]−316.0575315.0502315.05102.5300, 271Dmu
46Apigenin 7-O-glucuronideC21H18O1122.686** [M – H]−446.0875445.0802445.08020.2271, 253* Aau, Amu
473-MethoxysinensetinC21H22O830.813[M – H]−402.1301401.1228401.12341.5388, 373, 355, 327Eau
Flavanones
48Hesperetin 3′,7-O-diglucuronideC28H30O184.806[M – H]−654.1424653.1351653.13560.8477, 301, 286, 242* Babu, Cmbu, Debu, Eabu
49NarirutinC27H32O145.264[M – H]−580.1827579.1754579.17560.3271* Cmbu, Cabu, Cebu
50Hesperetin 3′-sulfateC16H14O9S7.389[M – H]−382.0354381.0281381.0277−1.0301, 286, 257, 242* Aethbu, Ambu, Babu, Cmbu, Dmbu, Eabu
51Hesperetin 3′-O-glucuronideC22H22O1213.741** [M – H]−478.1130477.1057477.10621.0301, 175, 113, 85* Eau, Emu
52Naringin 4′-O-glucosideC33H42O1932.747[M – H]−742.2295741.2222741.22321.3433, 271Cethbu
Flavonols
53Kaempferol 3-O-glucosyl-rhamnosyl-galactosideC33H40O204.719[M – H]−756.2135755.2062755.20751.7285Ceu
54Quercetin 3′-O-glucuronideC21H18O135.185[M – H]−478.0758477.0685477.0679−1.3301* Cabu, Bmu, Beu, Bethu, Bau
55Patuletin 3-O-glucosyl-(1->6)-[apiosyl(1->2)]-glucosideC33H40O225.616[M – H]−788.1982787.1909787.19191.3625, 463, 301, 271* Ceu, Cau
56Myricetin 3-O-arabinosideC20H18O127.182[M – H]−450.0821449.0748449.07500.4317* Cebu, Emu, Cau
57Quercetin 3-O-glucosyl-xylosideC26H28O1615.852[M – H]−596.1347595.1274595.12801.0265, 138, 116 *Eabu
58Isorhamnetin 3-O-glucuronideC22H20O1315.938[M – H]−492.0897491.0824491.0821−0.6315, 300, 272, 255Aabu`
59Myricetin 3-O-galactosideC21H20O1316.921** [M – H]−480.0911479.0838479.08482.1317* Eeu, Deu, Beu, Bau
60Myricetin 3-O-rhamnosideC21H20O1217.690[M – H]−464.0937463.0864463.08793.2317* Eeu, Beu
61Quercetin 3-O-arabinosideC20H18O1120.258** [M – H]−434.0850433.0777433.07964.4301* Eeu, Dmu, Dmbu
626-Hydroxyluteolin 7-rhamnosideC21H20O1120.989[M – H]−448.0991447.0918447.0904−3.1301* Eeu, Eau
63Quercetin 3′-sulfateC15H10O10S31.909[M – H]−381.9980380.9907380.9905−0.5301* Ceu, Cau, Beu, Bau, Aau
643-MethoxynobiletinC22H24O934.064[M + H]+432.1461433.1534433.15340.1403, 385, 373, 345* Aethu
Dihydroflavonols
65Dihydromyricetin 3-O-rhamnosideC21H22O1215.652** [M – H]−466.1112465.1039465.10430.9301* Eau, Deu
66DihydroquercetinC15H12O715.971[M – H]−304.0591303.0518303.05180.2285, 275, 151* Emu, Dabu
Dihydrochalcones
673-Hydroxyphloretin 2′-O-glucosideC21H24O114.593[M – H]−452.1354451.1281451.1280−0.2289, 273Bau
Isoflavonoids
686″-O-AcetylglycitinC24H24O115.896[M + H]+488.1333489.1406489.1398−1.6285, 270* Amu
692-Dehydro-O-desmethylangolensinC15H12O45.898[M – H]−256.0751255.0678255.06790.4135, 119* Cmbu, Babu
702′-HydroxyformononetinC16H12O55.934** [M – H]−284.0687283.0614283.06191.8270, 229* Emu, Eeu, Dmu
71ViolanoneC17H16O65.937[M – H]−316.0932315.0859315.0850−2.9300, 285, 135* Cmbu, Embu, Eeu
72SativanoneC17H16O516.707** [M – H]−300.0987299.0914299.09140.1284, 269, 225* Deu, Emu, Eau
73PseudobaptigeninC16H10O519.160** [M – H]−282.0506281.0433281.0431−0.7263, 237* Dau, Aau
74DalberginC16H12O421.505[M – H]−268.0717267.0644267.06564.5252, 224, 180* Deu, Ceu
756″-O-MalonyldaidzinC24H22O1222.473[M + H]+502.1085503.1158503.1149−1.8255Bau
76Genistein 4′,7-O-diglucuronideC27H26O1723.862** [M – H]−622.1174621.1101621.11223.4269Emu, Eeu, Dau, Cau, Bau, Aau
77GlycitinC22H22O1030.888[M – H]−446.1206445.1133445.1124−2.0285* Cethbu, Eabu
Other polyphenols
Hydroxycoumarins
78EsculetinC9H6O424.158[M – H]−178.0280177.0207177.02070.1149, 133, 89Bau
79ScopoletinC10H8O431.117[M – H]−192.0419191.0346191.03470.5176* Bethbu, Eeu, Eau, Bmu, Beu, Bau
Hydroxybenzoketones
802-Hydroxy-4-methoxyacetophenone 5-sulfateC9H10O7S24.081** [M – H]−262.0155261.0082261.0081−0.4181, 97* Aeu, Aau
Phenolic terpenes
81Carnosic acidC20H28O432.549** [M – H]−332.2004331.1931331.19330.6287, 269* Dethbu, Cethbu, Emu, Eau, Dmu, Deu, Dau, Ceu, Cethu, Cau, Bmu, Beu, Bethu, Bau, Amu, Aau
Tyrosols
823,4-DHPEA-ACC10H12O424.521[M – H]−196.0736195.0663195.0662−0.5135Amu
833,4-DHPEA-EDAC17H20O629.370[M – H]−320.1270319.1197319.1192−1.6275, 195* Aabu, Bmbu
Alkylmethoxyphenols
84EquolC15H14O316.915[M + H]+242.0943243.1016243.10191.2255, 211, 197* Emu, Eau, Dau
Other polyphenols
85Salvianolic acid CC26H20O1016.686[M – H]−492.1026491.0953491.09764.7311, 267, 249Embu
86ArbutinC12H16O719.621[M – H]−272.0901271.0828271.0827−0.4109* Ceu, Cau
Lignans
87Schisandrol BC23H28O73.062** [M – H]−416.1824415.1751415.1749−0.5224, 193, 165Aebu
887-HydroxymatairesinolC20H22O73.296** [M – H]−374.1381373.1308373.13110.8343, 313, 298, 285* Aebu, Eebu
89Todolactol AC20H24O716.242[M – H]−376.1546375.1473375.1469−1.1313, 137* Ambu, Dmbu
90SesaminC20H18O624.500** [M – H]−354.1138353.1065353.10680.8338, 163* Deu, Aabu, Babu
91ArctigeninC21H24O626.476[M – H]−372.1565371.1492371.14940.5356, 312, 295* Embu, Eebu, Dmu, Dau, Cau, Aeu, Aau
92Secoisolariciresinol-sesquilignanC30H38O1031.907[M – H]−558.2434557.2361557.23874.7539, 521, 509, 361Amu
Stilbenes
93ResveratrolC14H12O317.529** [M – H]−228.0787227.0714227.07171.3212, 185, 157, 143* Eau, Deu, Dabu
94Resveratrol 5-O-glucosideC20H22O833.742[M – H]−390.1283389.1210389.12141.0227Debu
* 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
Table 5. Characterization of phenolic compounds in conventional extracted seaweed samples by LC-ESI-QTOF-MS/MS.
Table 5. Characterization of phenolic compounds in conventional extracted seaweed samples by LC-ESI-QTOF-MS/MS.
No.Proposed CompoundsMolecular FormulaRT (min)Ionization (ESI+/ESI−)Molecular WeightTheoretical (m/z)Observed (m/z)Error
(ppm)		MS2 Product IonsSeaweed Samples
Phenolic acid
Hydroxybenzoic acids
1Protocatechuic acid 4-O-glucosideC13H16O93.062[M – H]−316.0818315.0745315.07511.9153* Cethbc, Cebc, Debc, Dmbc
24-Hydroxybenzoic acid 4-O-glucosideC13H16O87.687[M – H]−300.0839299.0766299.07732.3255, 137Dabc
34-HydroxybenzaldehydeC7H6O225.146[M – H]−122.0366121.0293121.02940.877* Bethbc, Cmbc, Dabc, Dmbc
42,3-Dihydroxybenzoic acidC7H6O425.793[M – H]−154.0259153.0186153.0185−0.7109Bethbc
52-Hydroxybenzoic acidC7H6O329.923[M – H]−138.0307137.0234137.02340.193Bethbc
6Gallic acidC7H6O531.949[M – H]−170.0209169.0136169.01402.4125* Aec, Aac, Bac, Bec, Bethc, Bmc, Cec, Cac, Cethc, Dec, Dethc, Dmc, Aabc, Debc
73-O-Methylgallic acidC8H8O533.658[M – H]−184.0355183.0282183.0280−1.1170, 142* Dec, Dethc, Dmc
Hydroxycinnamic acids
8Chlorogenic acidC16H18O93.078[M – H]−354.0969353.0896353.09021.7253, 190, 144* Cabc, Eabc
91-Sinapoyl-2,2′-diferuloylgentiobioseC43H48O213.119[M – H]−900.2677899.2604899.2574−3.3613, 201* Aebc, Debc
103-Feruloylquinic acidC17H20O94.687[M – H]−368.1081367.1008367.10121.1298, 288, 192,
191		Cmc
11Ferulic acid 4-O-glucosideC16H20O94.821[M – H]−356.1100355.1027355.10311.1193, 178, 149,
134		Eac
12Feruloyl tartaric acidC14H14O94.958[M – H]−326.0641325.0568325.0562−1.8193, 149* Dac, Dethc, Eac
13Caffeic acidC9H8O45.116[M – H]−180.0431179.0358179.03590.6143, 133 Emc
14Cinnamic acidC9H8O27.400** [M – H]−148.0523147.0450147.04510.7103* Cmbc, Ambc
15Caffeoyl glucoseC15H18O97.457[M – H]−342.0922341.0849341.0837−3.5179, 161Eebc
16p-Coumaric acid 4-O-glucosideC15H18O815.736[M – H]−326.0993325.0920325.09200.1163Bebc
173-p-Coumaroylquinic acidC16H18O816.364[M – H]−338.0974337.0901337.0900−0.3265, 173, 162Dabc
18p-Coumaroyl tartaric acidC13H12O816.591[M – H]−296.0522295.0449295.0437−4.1115Dmbc
19Hydroxycaffeic acidC9H8O517.250[M – H]−196.0376195.0303195.03040.5151* Aabc, Bethbc, Dabc, Dethbc
20Chicoric acidC22H18O1217.787[M – H]−474.0826473.0753473.0739−3.0293, 311Eac
21Rosmarinic acidC18H16O818.986[M – H]−360.0823359.0750359.07530.8179* Dmbc, Eabc, emc
22Cinnamoyl glucoseC15H18O720.687[M – H]−310.1035309.0962309.0955−2.3147, 131, 103* Dabc, Eabc
23Sinapic acidC11H12O522.780** [M – H]−224.0674223.0601223.0598−1.3205, 163* Cabc, Aabc, Ambc, Babc, Bebc, Bmbc, Cebc, Eabc, Eac
241,5-Dicaffeoylquinic acidC25H24O1223.930[M – H]−516.1262515.1189515.12134.7353, 335, 191,
179		Eec
25Caffeic acid 3-O-glucuronideC15H16O1024.346** [M – H]−356.0756355.0683355.0674−2.5179* Bmc, Bac, Dmc, Ambc, Dethbc
261,2,2′-TriferuloylgentiobioseC42H46O2030.541[M – H]−870.2536869.2463869.24862.6693, 517Aac
27m-Coumaric acidC9H8O332.821[M – H]−164.0477163.0404163.04082.5119Eethc
28Ferulic acid 4-O-glucuronideC16H18O1033.763[M + H]+370.0902371.0975371.0974−0.3193* Cebc, Dethbc
29p-Coumaroyl malic acidC13H12O734.094[M + H]+280.0580281.0653281.0650−1.1163, 119* Aebc, Ambc, Debc
Hydroxyphenylacetic acids
302-Hydroxy-2-phenylacetic acidC8H8O314.463[M – H]−152.0473151.0400151.04010.7136, 92* Cmbc, Aabc, Cabc, Dabc, Eabc, Eethc, Dmc, Eac
313,4-Dihydroxyphenylacetic acidC8H8O415.889[M – H]−168.0426167.0353167.03540.6149, 123* Aabc, Eethc
Hydroxyphenylpropanoic acids
32Dihydroferulic acid 4-O-glucuronideC16H20O103.064** [M – H]−372.1073371.1000371.10041.1195* Aabc, Aebc, Cabc, Cebc, Cmbc, Debc, Dmbc, Eabc, Aethbc, Dethbc
33Dihydrocaffeic acid 3-O-glucuronideC15H18O103.077** [M – H]−358.0905357.0832357.08330.3181* Aabc, Aebc, Eabc. Eebc, Aabc, Bac, Bec, Cmc
Flavonoids
Flavanols
34(+)-Gallocatechin 3-O-gallateC22H18O117.625[M – H]−458.0830457.0757457.0755−0.4305, 169* Babc, Bebc, Cebc, Debc
35Prodelphinidin dimer B3C30H26O1416.094[M + H]+610.1318611.1391611.1366−4.1469, 311, 291Aebc
36TheaflavinC29H24O1216.799[M – H]−564.1257563.1184563.11901.1545* Dac, Emc, Embc
37Procyanidin dimer B 1C30H26O1218.209** [M – H]−578.1444577.1371577.13781.2451* Cabc, Aebc, Dethbc
38(−)-EpigallocatechinC15H14O721.155[M – H]−306.0764305.0691305.06972.0261, 219* Aabc, Eabc
39(+)-Catechin 3-O-gallateC22H18O1024.152[M – H]−442.0876441.0803441.08162.9289, 169, 125Cac
40Theaflavin 3,3′-O-digallateC43H32O2026.891[M – H]−868.1512867.1439867.14390.1715, 563, 545Bac
414″-O-Methylepigallocatechin 3-O-gallateC23H20O1128.257[M – H]−472.1005471.0932471.0923−1.9169, 319* Cac, Bec, Debc
424′-O-Methyl-(−)-epigallocatechin 7-O-glucuronideC22H24O1334.052** [M – H]−496.1186495.1113495.11170.8451, 313* Bmc, Bethc, Dmc, Cac, Dmc, Emc, Embc
Flavones
43Apigenin 7-O-glucuronideC21H18O1120.616** [M + H]+446.0826445.0753445.07550.4271, 253* Cac, Dmc, Emc, Aac, Dabc
44Apigenin 7-O-(6″-malonyl-apiosyl-glucoside)C29H30O1720.668[M – H]−650.1509649.1436649.14451.4605* Dmc, Aac
45Apigenin 6-C-glucosideC21H20O1021.777[M – H]−432.1045431.0972431.09801.9413, 341, 311* Dethc, Eec
46Apigenin 7-O-apiosyl-glucosideC26H28O1422.663[M – H]−564.1524563.1451563.14591.4296Eec
473-MethoxysinensetinC21H22O830.907[M – H]−402.1278401.1205401.1202−0.7388, 373, 355, 327Dec
48Apigenin 6,8-di-C-glucosideC27H30O1532.223[M – H]−594.1592593.1519593.1509−1.7503, 473* Bac, Aec, Bec, Bethc
Flavanones
49Hesperetin 3′-sulfateC16H14O9S7.732[M – H]−382.0358381.0285381.0283−0.5301, 286, 257,
242		* Babc, Bebc, Cebc, Dmbc, Embc
50Hesperetin 3′-O-glucuronideC22H22O1216.771[M – H]−478.1134477.1061477.1055−1.3301, 175, 113,
85		* Emc, Dac, Dmc, Dabc, Eabc, Eebc
51Hesperetin 3′,7-O-diglucuronideC28H30O1818.725[M – H]−654.1415653.1342653.13562.1477, 301, 286,
242		Embc
52Naringin 4′-O-glucosideC33H42O1930.498[M – H]−742.2314741.2241741.22582.3433, 271Bec
53XanthohumolC21H22O531.272[M – H]−354.1486353.1413353.14253.4338, 309Aethc
Flavonols
54Kaempferol 3-O-glucosyl-rhamnosyl-galactosideC33H40O204.718[M – H]−756.2092755.2019755.2012−0.9285Cmc
55Kaempferol 3,7-O-diglucosideC27H30O1616.377[M – H]−610.1542609.1469609.14740.8447, 285Amc
56Isorhamnetin 3-O-glucuronideC22H20O1316.474[M – H]−492.0928491.0855491.08723.5315, 300, 272,
255		* Dac, Dmc
57Myricetin 3-O-rhamnosideC21H20O1217.258[M – H]−464.0946463.0873463.0867−1.3317* Dmc, Bac, Bec, Dmc, Eec, Emc
58Quercetin 3-O-arabinosideC20H18O1118.213[M – H]−434.0837433.0764433.07721.8301* Dac, Dmc
59Myricetin 3-O-arabinosideC20H18O1218.941[M – H]−450.0825449.0752449.0741−2.4317Dmc
606-Hydroxyluteolin 7-rhamnosideC21H20O1119.191[M – H]−448.1003447.0930447.09330.7301* Emc, Cac, Dac, Dethc, Eec, Emc, Cmbc, Eebc
61Quercetin 3-O-(6”-malonyl-glucoside)C24H22O1523.143[M – H]−550.0977549.0904549.0886−3.3303Bac
62Myricetin 3-O-galactosideC21H20O1324.356[M – H]−480.0909479.0836479.08370.2317* Emc, Bac, Bethc, Dmc
63Quercetin 3-O-glucosyl-xylosideC26H28O1625.144[M – H]−596.1402595.1329595.1313−2.7265, 138, 116Bac
64Quercetin 3′-O-glucuronideC21H18O1331.165[M – H]−478.0785477.0712477.07171.0301* Bec, Bac
65Quercetin 3′-sulfateC15H10O10S32.872[M – H]−382.0021380.9948380.99510.8301* Bmc, Aec, Bethc, Dec, Eac
66Quercetin 3-O-xylosyl-rutinosideC32H38O2033.752[M + H]−742.1933743.2006743.2003−0.4479, 317Debc
Dihydroflavonols
67Dihydroquercetin 3-O-rhamnosideC21H22O1119.734[M – H]−450.1147449.1074449.10770.7303* Eec, Dethc, Dmc, Emc, Eabc, Dabc, Eabc
68Dihydromyricetin 3-O-rhamnosideC21H22O1223.443[M – H]−466.1111465.1038465.10471.9301* Emc, Dac, Dethc, Eec
Dihydrochalcones
693-Hydroxyphloretin 2′-O-glucosideC21H24O114.663[M – H]−452.1348451.1275451.12770.4289, 273* Aac, Aec, Bec, Cac, Eec
Anthocyanins
70Delphinidin 3-O-glucosideC21H21O1226.489[M – H]−465.1016464.0943464.0940−0.6303Bac
71PelargonidinC15H11O532.266[M – H]−271.0618270.0545270.05574.4243, 197, 169, 141Eac
72Cyanidin 3,5-O-diglucosideC27H31O1632.278[M + H]+611.1632612.1705612.17111.0449, 287* Aethbc, Bebc, Debc
73Peonidin 3-O-diglucoside-5-O-glucosideC34H43O2133.383[M – H]−787.2331786.2258786.2246−1.5625, 478, 317* Cethc, Dec
Isoflavonoids
746″-O-AcetyldaidzinC23H22O104.703[M – H]−458.1218457.1145457.1139−1.3221Bmc
755,6,7,3′,4′-PentahydroxyisoflavoneC15H10O75.266[M – H]−302.0439301.0366301.03763.3285, 257Amc
762′,7-Dihydroxy-4′,5′-dimethoxyisoflavoneC17H14O66.882[M – H]−314.0767313.0694313.06981.3300, 282* Eac, Eec
776″-O-MalonylgenistinC24H22O1314.363** [M + H]+518.1069517.0996517.09970.2271* Eac, Eac
782-Dehydro-O-desmethylangolensinC15H12O416.548[M – H]−256.0746255.0673255.0667−2.4135, 119* Emc, Bmc
79SativanoneC17H16O516.563** [M – H]−300.0994299.0921299.09230.7284, 269, 225* Aec, Aac, Amc, Bec, Cac, Emc, Ambc
806″-O-MalonylglycitinC25H24O1318.919[M – H]−532.1215531.1142531.11450.6285, 270, 253* Emc, Eec
81Formononetin 7-O-glucuronideC22H20O1021.977[M – H]−444.1048443.0975443.0972−0.7267, 252* Cac, Eac
822′-HydroxyformononetinC16H12O522.421[M + H]+284.0694285.0767285.0764−1.1270, 229* Dmc, Cac, Emc
83ViolanoneC17H16O624.296[M – H]−316.0921315.0848315.0842−1.9300, 285, 135* Aebc, Ambc
84Genistein 4′,7-O-diglucuronideC27H26O1725.445[M – H]−622.1114621.1041621.10674.2269* Aac, Bec, Dethc, Debc
856″-O-MalonyldaidzinC24H22O1233.752** [M + H]+502.1093503.1166503.11782.4255* Debc, Dabc, Embc, Dac, Cac, Eac, Emc
Other polyphenols
Hydroxycoumarins
86EsculetinC9H6O424.191[M – H]−178.0248177.0175177.0171−2.3149, 133, 89Bec
87ScopoletinC10H8O431.153[M – H]−192.0407191.0334191.03350.5176* Babc, Aebc, Bebc, Bethbc, Bmbc, Dabc, Debc, Dmbc, Dmc, Bec, Bmc, Cac, Eec, Emc
Hydroxybenzaldehydes
88p-AnisaldehydeC8H8O230.932[M – H]−136.0516135.0443135.0442−0.7122, 109* Bethc, Eac, Eec, Eethc, Emc
893-Hydroxy-3-(3-hydroxyphenyl)
propionic acid		C9H10O432.498[M – H]−182.0582181.0509181.0507−1.1163, 135, 119Eac
Phenolic terpenes
90Carnosic acidC20H28O432.545[M – H]−332.1985331.1912331.1906−1.8287, 269* Bac, Aac, Aec, Aethc, Amc, Bac, Bec, Bethc, Bmc, Cec, Dac, Dethc, Eec, Emc, Dethbc, Eabc
Tyrosols
913,4-DHPEA-ACC10H12O44.682[M – H]−196.0727195.0654195.06540.1135Cac
92Hydroxytyrosol 4-O-glucosideC14H20O816.175[M – H]−316.1149315.1076315.10760.1153, 123Cmbc
933,4-DHPEA-EDAC17H20O628.718[M – H]−320.1280319.1207319.12090.6275, 195Bethbc
Alkylmethoxyphenols
94EquolC15H14O318.110** [M + H]+242.0943243.1016243.1014−0.8255, 211, 197* Cabc, Amc, Aac, Aec, Eac, Eethc
Other polyphenols
95Salvianolic acid BC36H30O1616.693[M – H]−718.1517717.1444717.1421−3.2519, 339, 321,
295		Embc
96Lithospermic acidC27H22O1217.324** [M – H]−538.1097537.1024537.1018−1.1493, 339, 295* Eabc, Cabc, Cethbc, Dmc, Cec
97Salvianolic acid CC26H20O1021.929[M – H]−492.1052491.0979491.09892.0311, 267, 249* Debc, Dmbc
Lignans
98Todolactol AC20H24O716.368[M – H]−376.1539375.1466375.14701.1313, 137* Debc, Embc
997-HydroxymatairesinolC20H22O728.718** [M – H]−374.1379373.1306373.13111.3343, 313, 298,
285		* Bethbc, Dabc, Eebc, aac
100ArctigeninC21H24O630.493[M – H]−372.1565371.1492371.1475−4.6356, 312, 295Aethc
101Secoisolariciresinol-sesquilignanC30H38O1030.674[M – H]−558.2463557.2390557.23900.1539, 521, 509,
361		Aec, debc
1027-OxomatairesinolC20H20O732.347[M + H]+372.1184373.1257373.1256−0.3358, 343, 328,
325		* Babc, Bebc, Bethbc, Cmbc
103SesaminC20H18O632.442[M – H]−354.1124353.1051353.10612.8338, 163Eec
Stilbenes
104Resveratrol 5-O-glucosideC20H22O816.382[M – H]−390.1310389.1237389.1234−0.8227Embc
* 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.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Subbiah, V.; Ebrahimi, F.; Agar, O.T.; Dunshea, F.R.; Barrow, C.J.; Suleria, H.A.R. Comparative Study on the Effect of Phenolics and Their Antioxidant Potential of Freeze-Dried Australian Beach-Cast Seaweed Species upon Different Extraction Methodologies. Pharmaceuticals 2023, 16, 773. https://doi.org/10.3390/ph16050773

AMA Style

Subbiah V, Ebrahimi F, Agar OT, Dunshea FR, Barrow CJ, Suleria HAR. Comparative Study on the Effect of Phenolics and Their Antioxidant Potential of Freeze-Dried Australian Beach-Cast Seaweed Species upon Different Extraction Methodologies. Pharmaceuticals. 2023; 16(5):773. https://doi.org/10.3390/ph16050773

Chicago/Turabian Style

Subbiah, Vigasini, Faezeh Ebrahimi, Osman T. Agar, Frank R. Dunshea, Colin J. Barrow, and Hafiz A. R. Suleria. 2023. "Comparative Study on the Effect of Phenolics and Their Antioxidant Potential of Freeze-Dried Australian Beach-Cast Seaweed Species upon Different Extraction Methodologies" Pharmaceuticals 16, no. 5: 773. https://doi.org/10.3390/ph16050773

APA Style

Subbiah, V., Ebrahimi, F., Agar, O. T., Dunshea, F. R., Barrow, C. J., & Suleria, H. A. R. (2023). Comparative Study on the Effect of Phenolics and Their Antioxidant Potential of Freeze-Dried Australian Beach-Cast Seaweed Species upon Different Extraction Methodologies. Pharmaceuticals, 16(5), 773. https://doi.org/10.3390/ph16050773

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop