Process Optimization for Polyphenol Extraction from Macroalgae Residues and Assessment of Their Compositions, Antioxidant Activities, and Glycosidase Inhibition

Abstract

Macroalgae are often used to produce sodium alginate, but their by-products have not been fully utilized. This study aimed to optimize the extraction of bound polyphenols (BPs) from Macrocystis pyrifera (L.) residues, analyze the composition of free polyphenols (FPs) and BPs, and evaluate their antioxidant activities and ability to inhibit glycosidase activity. The optimal conditions for extracting BPs achieved by the response surface method were as follows: 50 °C, a solid–liquid ratio of 1:50, an alkaline hydrolysis time of 2.38 h, and a NaOH concentration of 8 mol/L. Polyphenol content determination results indicated that FPs had significantly higher total polyphenols (13.02 ± 0.05 μg GAE/mg) and phlorotannin (3.44 ± 0.04 μg PE/mg) than BPs (6.57 ± 0.07 μg GAE/mg and 1.32 ± 0.20 μg PE/mg). HPLC/ESI-QTOF-MS showed distinct profiles: FPs had one polyhydroxy phenol, nine flavonoids, and four additional compounds, whereas BPs had five flavonoids and four other compounds. Antioxidant activity was found to be higher in FPs than in BPs (DPPH: 3.03 vs. 1.79 μg TE/mg; FRAP: 19.40 vs. 7.43 μg TE/mg). Furthermore, FPs exhibited 4.59- and 11-fold higher inhibition capacity toward α-amylase and α-glucosidase, respectively, compared to BPs. The results provide valuable basic data for the application of macroalgae residues in the marine biological industry and reveal their potential hypoglycemic ability.

1. Introduction

Polyphenols are the most significant group of compounds that determine the biomedical importance of marine algae [1]. Algae have been demonstrated to contain large amounts of polyphenols [2], which exhibit various bioactive properties, including antitumor [3], anti-inflammatory [4], and anti-diabetic [5] effects. These findings underscore the necessity for more comprehensive and multidimensional investigations into the polyphenolic compounds in algae. Polyphenols are divided into free polyphenols (FPs) and bound polyphenols (BPs) according to solubility and extraction methods [6]. The extraction of BPs is usually more complicated than that of FPs, often requiring chemical or enzymatic hydrolysis [7,8]. There is research showing the potential antioxidant and anti-inflammatory activities of BPs in algae, which are beneficial to human health [9]. Consequently, optimizing extraction techniques for BPs in algae warrants systematic investigation to fully exploit their bioactive potential.

Macroalgae are aquatic photosynthetic organisms that are mainly classified as green, red, or brown algae [10,11]. Macrocystis pyrifera (L.) Ag. is an alga of the genus Macrocystis under the family Laminariaceae, which is part of the order Laminariales within the phylum Heterokontophyta [12]. Macroalgae are rich in nutrients such as flavonoids, tannins, and polysaccharides, which are often used to extract sodium alginate. Sodium alginate is a macroalgae extract that exhibits significant antioxidant activity and has broad application prospects [13]. Research has shown that sodium alginate helps prevent diseases such as cardiovascular disease, diabetes, and non-alcoholic fatty liver disease (NAFLD) due to its richness in bioactive compounds like polyphenols and flavonoids [14,15]. Beyond their ecological roles, macroalgae are commercially utilized in food processing and agricultural applications [16,17]. However, studies have shown that about 30–50% of the raw materials in the macroalgae industry eventually become residues [18]. As a by-product of the extraction process of sodium alginate, macroalgae residue is mainly used for feed, fertilizers, and biological refining [19], as it is still rich in polyphenols, dietary fiber, and other active components, which have not been fully utilized. There is a significant research gap regarding the methodological optimization of macrocystis residue extraction and subsequent analysis of their biological properties.

The hyperglycemia potential of algal polyphenols has been extensively investigated [5], while studies on the bioactivity of polyphenols from macroalgae residues currently remain limited. Investigating the inhibitory activities of α-amylase and α-glucosidase from macroalgae residues will facilitate future exploration of their potential beneficial effects on diabetes management. The present study focuses on achieving the reuse of active ingredients in the macroalgae residue by optimizing the extraction of polyphenols from these residues, investigating their composition via high-performance liquid chromatography–electrospray ionization–quadrupole time-of-flight tandem mass spectrometry (HPLC-ESI-QTOF-MS/MS), and evaluating their antioxidant activity and ability to inhibit glycosidase, thereby uncovering the potential uses of macroalgae residue as a source of natural antioxidants that are suitable for functional food formulations or pharmaceutical products aimed at hyperglycemia management.

2. Materials and Methods

2.1. Materials and Chemicals

2.1.1. Samples

The macroalgae residue obtained after alginate extraction was derived from Macrocystis pyrifera (provided by Qingdao Mingyue Seaweeds Group Co., Ltd., Qingdao, Shandong, China). The residue was dried at 55 °C, crushed into a powder by a grinder, and sieved through a 60-mesh sieve.

2.1.2. Reagents and Equipment

Folin’s phenol reagent (1 mol/L), 3,5-Dinitrosalicylic acid (DNS), α-amylase (3.7 U/mg, Bacillus), and 1,1-diphenyl-2-trinitrophenylhydrazine (≥98%) were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). Formic acid (98.0%) was purchased from Sigma-Aldrich. Gallic acid (99%), resorcinol (>99.0%), 2,4-dimethoxybenzaldehyde (DMBA) (98%), and pNPG (99%) were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). 2,4,6-Tripyridyl-s-triazine (98%) and a-glucosidase (33 U/mg, Saccharomyces) were purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). 4-Nitrophenyl octanoate (90%) was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

2.2. Extraction of FPs

The approach used in this study is illustrated in Figure 1. FPs were obtained via extraction with ethanol and water. The conditions for extracting free phenols from the macroalgae residue include an ethanol volume fraction of 85%, a solid–liquid ratio of 1:10, and magnetic stirring at 55 °C for 6 h. After extraction and filtration, we repeated the extraction process with ethanol twice and retained the filtrate. We then dried the filter residue at 55 °C and stored it.

2.3. Single-Factor Extraction Experiments for BPs

The extraction process for BPs was optimized using alkaline hydrolysis followed by ethanol precipitation. Firstly, the Macrocystis pyrifera residue was added to a specified volume of NaOH solution at a predetermined concentration, and the mixture was hydrolyzed at a controlled temperature. Subsequently, two volumes of 95% ethanol were added, and extraction was carried out for 1 h with continuous shaking. The extract was then centrifuged at 1118× g for 15 min, and the supernatant was collected for bound polyphenol quantification. Taking the rate of BP extraction as the outcome, we fixed other parameters and investigated the effects of four factors: temperature (30, 40, 50, 60, 70, 80, and 90 °C); NaOH concentration (2, 4, 6, 8, and 10 mol/L); alkaline hydrolysis time (1, 2, 3, 4, and 5 h); and liquid–solid ratio (1:10, 1:20, 1:30, 1:40, and 1:50), The specific experimental methods are shown in

Table 1. Single-factor experimental scheme.

No.	Liquid–Solid Ratio	NaOH Concentration (mol/L)	Alkaline Hydrolysis Time (h)	Alkaline Hydrolysis Temperature (°C)
1	1:40	4	1	30, 40, 50, 60, 70, 80, 90
2	1:40	2, 4, 6, 8, 10	1	50
3	1:40	4	1, 2, 3, 4, 5	50
4	1:10, 1:20, 1:30, 1:40, 1:50	4	2	50

.

2.4. Optimization of Extraction Conditions and Quantification of Total Phenolic Contents

The optimal method for extracting BPs from the macroalgae residue was determined via the Box–Behnken response surface method (RSM). A three-factor and three-plane heart cube design was used, including 16 experimental runs. The NaOH concentration (A, mol/L), extraction time (B, h) and liquid–solid ratio (C) at 50 °C were altered, and the response variable was the polyphenol content (Y). The specific experimental scheme and results are shown in

Table 2. Factors and levels of the RSM.

Level	A: NaOH Concentration (mol/L)	B: Alkaline Hydrolysis (h)	C: Liquid–Solid Ratio
−1	4	1	1:30
0	6	2	1:40
1	8	3	1:50

.

The total phenol and phlorotannin (phloroglucinol as the standard) contents of the samples extracted under the optimized conditions were determined simultaneously. The total phenol content (TPC) test method was a modified version of the Folin–Ciocalteu method, and the DMBA assay was modified from the phlorotannin content test [20]. Given the thermolability of polyphenolic antioxidant activity, single-factor tests additionally incorporated temperature effects. The experimental data was analyzed using Design Expert software (version 8.0.6).

2.5. Qualitative Analysis of Phenolic Compounds by HPLC-ESI-QTOF-MS/MS

Samples were dissolved in 100 μL of methanol and centrifuged at 1610× g and 4 °C for 10 min using a Microfuge 22R Centrifuge (Beckman Coulter, Brea, CA, USA). Then, the supernatant was extracted and used in the subsequent steps.

Capillary HPLC (Ultimate 3000, Thermo Fisher, Waltham, MA, USA) with a C18 column was used for gradient elution, with the mass spectrometer (TripleTOF5600 +, AB SCIEX, Redwood, CA, USA) in positive/negative ion mode. A Sepax GP-C18 (1.8 µm, 120Å, 2.1 mm×150 mm) column (Sepax Technologies, Newark, DE, USA) was employed for separation. The flow rate employed was 0.3 mL/min and the column was held at a constant temperature of 40 °C. The gradient elution parameters were as follows: 0~10 min with 5% B, 10~17 min with 70% B, 17~18 min with 100% B, 18~19 min with 100% B, and 19~21 min with 5% B. The ESI source was operated in positive and negative ion modes. The data were imported into MS-DIAL 4.70 software for data processing and compared with data from three libraries: MassBank, Respect, and GNPS (14,951 records in total).

2.6. Antioxidant Activity and Glycosidase Activity Inhibition Assay

2.6.1. Antioxidant Activity Assay

The antioxidant activity of free and bound polyphenol extracts was evaluated via the DPPH and FRAP methods, respectively. The antiradical activity of DPPH was determined using an assay, as explained elsewhere [21,22]. Aliquots of 0.5 mL of Trolox standard solutions at varying concentrations were transferred to 1.5 mL microcentrifuge tubes, followed by the addition of 0.5 mL of a freshly prepared 0.2 mmol/L DPPH methanolic solution. After mixing, the reaction was carried out for 0.5 h in the dark, and absorbance was measured at 520 nm. All samples, blanks, and controls were prepared in triplicate. The radical scavenging activity of DPPH was calculated as follows:

Scavenging rate (%) = [(A_c − A_n + A_b)/A_c] × 100%

(1)

The FRAP method was modified from Rumpf et al.’s work [23]. A total of 0.05 mL of the Trolox gradient solution was added to 1.45 mL of the FRAP working solution. The reaction was carried out in a water bath at 37 °C for 10 min, and absorbance was measured at 593 nm with a microplate reader. All samples, blanks, and reference substances were prepared in parallel and in triplicate. From the Trolox calibration curve, the results of the antioxidant activity assays were expressed as the concentration in Trolox equivalent (TE), and the calculation formula was as follows:

TE = (A_n/A_T) × C_T

(2)

2.6.2. Glycosidase Activity Inhibition Assay

Both FPs and BPs extracts inhibited the activities of α-glucosidase and α-amylase The method for determining the inhibitory activity of α-amylase was modified from Liu et al.’s study [24]. The diluted free and bound polyphenol extracts were added to different tubes with either 0.05 mL of the α-amylase solution (0.1 U/mL), 0.1 M pH 6.8 PBS, or 0.05 mL of an inhibitor, and then shaken at 37 °C for 15 min. Then, 0.1 mL of a starch solution (1%) was added to each tube and the reaction was carried out at 50 °C for 10 min. Each tube was supplemented with 0.4 mL of a DNS solution in a boiling water bath for 10 min, and then cooled to room temperature. Then, 1 mL of water was added to each tube, and the absorbance at 540 nm was measured. All samples, blanks, and reference substances were prepared in parallel and in triplicate. The inhibition rate was calculated using Formula (3) [25].

Inhibition rate (%) = [1 − (OD_A − OD_a)/(OD_B − OD_b)] × 100

(3)

The methodology for assessing the inhibitory activity of α-glucosidase has been described by Yang et al. [26]. First, we added 0.1 M phosphate buffer, an inhibitor, and α-glucosidase to the experimental group, the control group, and the blank group according to the protocol. The mixture was incubated in a water bath at 37 °C for 10 min. Subsequently, 0.1 mL of pNPG (5 mmol/L) was added to each group, and the samples were incubated at 37 °C for 1 h. Then, 1 mL of a 1 mol/L sodium carbonate solution was added. All samples, blanks, and controls were prepared in parallel and in triplicate. Absorbance was measured at 405 nm. The formula for calculating the inhibition rate is shown in Formula (3).

2.7. Statistical Analysis

Three separate extraction experiments were performed to determine the results, which are presented as the mean ± standard deviation. SPSS 16.0 software was used to analyze the data, and one-way ANOVA followed by the LSD (least significant difference) mean test was applied. A p-value of p < 0.05 was considered statistically significant.

3. Results and Discussion

3.1. Quantification of Free Polyphenols from the Macroalgae Residue

Through extraction and assays, it was determined that the crude extract yield of FPs from the macroalgae residue was 4.5%. The polyphenol content was 13.02 ± 0.07 μg GAE/mg and 3.45 ± 0.04 μg PE/mg. In contrast, the rate of BP extraction varied under different experimental conditions.

3.2. Optimized Conditions for Bound Polyphenols

In the single-factor experiments, the influence of various factors on the extraction rate and antioxidant activity of BPs from macroalgae residue was evaluated. The design and results of the Box–Behnken experiments are shown in

Table 3. The design and results of the Box–Behnken experiments.

No.	NaOH Concentration	Alkaline Hydrolysis Time (B)	Liquid–Solid Ratio (C)	Total Polyphenol Content (Y)
1	4 (−1)	1 (−1)	40 (0)	5.27
2	8 (1)	1 (−1)	40 (0)	6.69
3	4 (−1)	3 (1)	40 (0)	7.18
4	8 (1)	3 (1)	40 (0)	8.88
5	4 (−1)	2 (0)	30 (−1)	5.98
6	8 (1)	2 (0)	30 (−1)	7.72
7	4 (−1)	2 (0)	50 (1)	7.38
8	8 (1)	2 (0)	50 (1)	9.38
9	6 (0)	1 (−1)	30 (−1)	3.57
10	6 (0)	3 (1)	30 (−1)	7.20
11	6 (0)	1 (−1)	50 (1)	6.94
12	6 (0)	3 (1)	50 (1)	8.35
13	6 (0)	2 (0)	40 (0)	7.49
14	6 (0)	2 (0)	40 (0)	7.67
15	6 (0)	2 (0)	40 (0)	7.80
16	6 (0)	2 (0)	40 (0)	7.60

. As shown in Figure 2A, the efficiency of BP extraction exhibited a positive correlation with the alkaline hydrolysis temperature from 30 °C to 90 °C. This enhancement is attributed to increased molecular mobility and accelerated solvent diffusion at higher temperatures, which reduce viscosity and improve the solubility and extraction yield of polyphenols [27]. The extract maintained high antioxidant activity within the temperature range of 30 °C to 50 °C, beyond which a continuous decline was observed from 60 °C to 90 °C. The optimal extraction effect was observed at an alkali hydrolysis temperature of 50 °C. Additionally, according to Figure 2B–D, the optimal extraction conditions for BPs were a solid–liquid ratio of 1:40, a NaOH concentration of 6 mol/L, and an alkaline hydrolysis time of 2 h.

As evidenced by the ANOVA results presented in

Table 4. Variance analysis of the model.

Source	Sum of Squares	Degree of Freedom	Mean Square	F-Value	p-Value	Significant
Model	28.27	9	3.14	39.54	0.0001	**
A	5.87	1	5.87	73.86	0.0001	**
B	10.44	1	10.44	131.48	<0.0001	**
C	7.19	1	7.19	90.54	<0.0001	**
AB	0.019	1	0.019	0.24	0.6409
AC	0.016	1	0.016	0.21	0.6646
BC	1.23	1	1.23	15.47	0.0077	**
A2	0.22	1	0.22	2.73	0.1496	*
B2	3.02	1	3.02	37.98	0.0008	**
C2	0.26	1	0.26	3.33	0.1178
Residual	0.48	6	0.079
Lack of fit	0.43	3	0.14	8.67	0.0547
Pure error	0.049	3	0.016
Cor. total	28.74	15

Note: ** indicates p < 0.01, which means a highly significant difference; * indicates p < 0.05, which means a significant difference.

, the established model demonstrates statistical significance (p < 0.05), confirming its validity. The significance of the factors on the response value was determined to decrease in the order C > B > A. The lack-of-fit test yielded a p-value of 0.0547, indicating that the lack-of-fit error is negligible compared to the pure error. The regression model, representing the amount of BPs extracted from macroalgae residues as a function of the NaOH concentration (Figure 3A), extraction time (B), and solid-to-liquid ratio (C), is given by the following equation:

Y = −9.75 − 0.47A + 6.63B − 0.39C + 0.035AB − (3.21119E − 003) AC − 0.05541BC + 0.058A² − 0.87B² − (2.57172E − 003) C²

(4)

The steepness of the slope in the response surface 3D plot reflects the strength of the interaction between the two factors; a steeper slope indicates a stronger influence on the response value, while a gentler slope suggests a weaker effect [28]. Four parallel experiments were carried out. The optimal extraction rate was achieved with a solid–liquid ratio of 1:50, alkaline hydrolysis time of 2.38 h and a NaOH concentration of 8 mol/L at 50 °C. The purity of BPs was 9.34 μg GAE/mg, and 97.19% of the predicted value could be achieved.

After extracting FPs, BPs were extracted under the optimal extraction conditions. The BP content was 6.51 ± 0.45 μg GAE/mg and 1.32 ± 0.20 μg PE/mg.

3.3. Characterization of the Two Polyphenol Extracts

Characterization of Polyphenol Extracts by HPLC-ESI-QTOF-MS/MS and Comparison with MSn Data from an Online Database

The total ion chromatograms (TICs) of FPs and BPs, obtained from the HPLC analysis, are shown in Figure 4 and Figure 5, respectively. The TIC profiles display well-resolved peaks, indicating the presence of multiple compounds in the sample. FPs were predominantly ionized in negative mode, whereas BPs were ionized in both positive and negative modes.

In this study, FPs and BPs from the macroalgae residue were analyzed using HPLC-ESI-QTOF-MS/MS and the retention times (Rt), formulae, and accurate mass spectral data were compared with online databases and the literature (

Table 5. Characterization of polyphenol extracts via HPLC-ESI-QTOF-MS/MS and comparison with MSn data from an online database.

Identity	Formula	Retention Time (min)	Addition Ion	m/z	Reference (m/z)	Peak
Free Polyphenols
Polyhydroxybenzene
5-{8(Z),11(Z)-pentadecadienyl}resorcinol	C21H32O2	14.0939	[M−H]−	315.3153	315.3175	F′8
Flavonoids
6,8-dihydroxy-2,2,4,4-tetramethyl-7-(3-methylbutanoyl)-9-(2-methylpropyl)-9H-xanthene-1,3-dione	C26H34O6	20.41	[M−H]−	441.3863	441.3867	F′15
5-O-methylgenistein	C16H12O5	16.68	[M−H]−	283.3203	283.3212	F′11
Epiafzelechin (2R,3R)(-)	C15H14O5	9.148	[M−H]−	273.1768	273.1747	F′2
5-hydroxy-2′,4′,7,8-tetramethoxyflavone	C19H18O7	20.18	[M−H]−	339.3171	339.3141	F′14
7,4′-dimethoxysoflavone	C17H14O4	20.65	[M−H]−	281.3031	281.2998	F′16
Catechin	C15H14O6	1.25	[M−H]−	289.127	289.1219	F′1
3-[(Z)-heptadec-10-enyl]benzene-1,2-diol	C23H38O2	15.65	[M−H]−	345.2745	345.2799	F′10
Neobavaisoflavone	C20H18O4	10.64	[M+H]+	345.1133	345.11	F3
2′′-O-rhamnosyl icariside II	C33H40O14	15.4	[M+H]+	683.2292	683.23	F12
Others
Fraxin	C16H18O10	14.82	[M+H]+	409.0553	409.0532	F10
α-Tochopherol	C29H50O2	15.33	[M−H]−	429.3738	429.3738	F′9
Esculin	C15H16O9	1.4	[M+H]+	363.0636	363.06	F1
2-hydroxy-6-pentadecylbenzoic acid	C22H36O3	13.53	[M−H]−	347.2638	347.258	F′6
Bound polyphenols
Flavonoids
2′,5′-dihydroxy-4-methoxychalcone	C16H14O4	8.52	[M−H]−	269.1664	269.1674	B′5
5-O-methylgenistein	C16H12O5	17.35	[M−H]−	283.3191	283.3212	B′6
Quercetin 3,7-dimethyl ether	C17H14O7	1.88	[M−H]−	329.0576	329.062	B′7
Licoflavone A	C20H18O4	10.69	[M+Na]+	345.1138	345.11	B3
Licoricidin	C26H32O5	20.79	[M+H]+	447.2125	447.21	B7
Others
Juglone	C10H6O3	5.03	[M+H]+	212.9939	212.9949	B1
Alpha-estradiol	C18H24O2	5.58	[M+H]+	255.1714	255.1744	B2
3-methoxy-2-(3-methylbut-2-enyl)-5-pentylphenol	C17H26O2	6.15	[M−H]−	261.1752	261.1783	B′3
Atalaphylline	C23H25NO4	7.28	[M−H]−	378.2395	378.2336	B′4

). The FPs contained 14 identifiable compounds, including 1 polyhydroxy phenol, 9 flavonoids, and 4 other phenolic substances. In contrast, the BPs comprised nine compounds, consisting of five flavonoids and four other phenolic components. FPs contain more flavonoids and other substances than BPs. This difference may be attributed to the decomposition of some heat-sensitive flavonoids caused by the alkaline hydrolysis conditions in the BP extraction process [29]. The phlorotannin components in FPs and BPs were identified by matching data from the literature (

Table 6. Characterization of polyphenol extracts via by HPLC-ESI-QTOF-MS/MS and comparison with MSn data from the literature.

Identity	Addition Ion	Measured Mass (m/z)	MS/MS Fragment Detected (m/z)	Algae Source	Peak
Free polyphenols
Fuhalols
2-(1)	[M+H]+	267.2	141	Sargassum fusiforme	F5
3-(1)	[M+H]+	391.3	125	Sargassum fusiforme	F6
5-(1)	[M+H]+	639.3	388	Sargassum fusiforme	F17
5-(2)	[M+H]+	655.3	527, 389	Sargassum fusiforme	F8
6-(3)	[M+H]+	795.3	667	Sargassum fusiforme	F14
Dihydroxytetrafuhalol	[M+H]+	547.4	529	Sargassum muticum	F11
Dihydroxypentafuhalol	[M+H]+	671.3	625, 623, 607, 527, 465, 402, 341, and 263	Sargassum muticum	F4
Dihydroxyhexafuhalol	[M+H]+	795.3	777, 749, 731, 653, and 465	Sargassum muticum	F14
Pentafuhalol	[M+H]+	639.3	621, 513, 385, and 373	Sargassum muticum	F15
Hydroxytetrafuhalol	[M+H]+	655.3	637, 527, and 389	Sargassum muticum	F8
Bifuhalol trimer	[M+H]+	795.3	515	Carpophyllum flexuosum	F14
Octafuhalol	[M+H]+	1011.7	621	Sargassum muticum	F7
nd	[M+H]+	653.3	607 and 465	Laminaria digitata	F16
nd	[M+H]+	653.3	607 and 465	Laminaria digitata	F18
Dihydroxypentafuhalol	[M−H]−	669.6	625	Sargassum muticum	F′13
Dihydroxyhexafuhalol	[M−H]−	793.6	465	Sargassum muticum	F′12
Hydroxytrifuhalol	[M−H]−	405.2	154	Carpophyllum flexuosum	F′3
Eckols
3-(1)	[M+H]+	389.2	329, 245, and 123	Sargassum fusiforme	F2
Eckol	[M−H]−	371.2	335	Sargassum fusiforme	F′7
Eckol derivative	[M−H]−	401.3	371	Fucus vesiculosus	F′5
Others
Tetramer	[M+H]+	499.4	481 and 233	Fucus vesiculosus	F9
Fucol	[M+H]+	623.3	495, 479, 461, 373, and 355	Laminaria digitata	F13
Pentaphlorethol	[M+H]+	623.3	603 and 493	Sargassum muticum	F19
Pentamer	[M+H]+	623.3	605, 497, 434, and 356	Fucus vesiculosus	F13
Phlorethol	[M−H]−	745.5	477	Laminaria digitata	F′4
Bound polyphenols
Fuhalols
3-(1)	[M+H]+	391.3	123	Sargassum fusiforme	B5
6-(3)	[M+H]+	795.5	667 and 389	Sargassum fusiforme	B6
Dihydroxyhexafuhalol	[M+H]+	795.5	777, 749, 731, 513, 511, 485, 483, and 385	Sargassum muticum	B6
Trifuhalol	[M+H]+	391.3	251	Sargassum muticum	B5
Bifuhalol trimer	[M+H]+	795.5	515 and 261	Carpophyllum flexuosum	B6
Eckols
6-(3)	[M+H]+	793.5	747 and 385	Sargassum fusiforme	B4
2-(1)	[M−H]−	263.2	245 and 219	Sargassum fusiforme	B′2
Others
Fucol	[M+H]+	623.3	477 and 371	Fucus vesiculosus	B4
Fucophlorethol	[M+H]+	623.3	461, 373, and 355	Fucus vesiculosus	B4
Pentaphlorethol	[M+H]+	623.3	493	Sargassum muticum	B4
5	[M+H]+	623.3	605, 373, 356, 355, and 340	Sargassum muticum	B4

). Fuhalols and eckols were identified in both FPs and BPs, but more phlorotannin species were detected in FPs. These compounds are easily detected in positive ion mode, while phenolic acids are more easily detected in negative ion mode. It may be that phlorethols were not detected in BPs because the polyhydroxy phenol group in the BPs formed an ether bond with the cell wall polysaccharide, shielding the ionization site and resulting in blocked negative ionization [30]. A comparison with published mass spectrometry data on fucoidan polyphenols showed that the polyphenols extracted from macroalgae residues were most similar to those found in Sargassum fusiforme, Sargassum muticum, Laminaria digitata, and Fucus vesiculosus.

3.4. Antioxidant Activity and Inhibitory Effect on Glycosidase Activity

Table 7. Comparison chart of antioxidant and enzyme inhibitory activity.

Polyphenol Type	DPPH (μg TE/mg)	FRAP (μg TE/mg)	Anti-Amylase (mg AE/mg)	Anti-Glucosidase (μg AE/g)
Free	3.03	19.40	0.11	21.53
Bound	1.79	7.43	0.01	0.99

shows the differences in antioxidant capacity and glycosidase inhibition between the two polyphenols. The DPPH scavenging activity assay on the macroalgae residue revealed that the antioxidant capacity in FPs was higher (3.03 μg TE/mg) than that in BPs (1.79 μg TE/mg). The FRAP test results showed a consistent trend (FPs: 1.49 μg TE/mg vs. BPs: 1.14 μg TE/mg) in that the macroalgae residue’s antioxidant capacity is higher than that of the macroalgae edulis (0.66 ± 0.02 μg TE/mg) [31]. The high content of phenolic compounds and flavonoids was considered to be an important cause of antioxidant capacity in previous studies [32]. Therefore, FPs may exhibit better FRAP antioxidant capacity due to their high contents of flavonoid species and unique polyhydroxy phenols. The DPPH assay measures the free radical scavenging ability using the electron-providing ability, while the FRAP assay evaluates the reducing ability via electron transfer [33].

The inhibitory activity of polyphenols against α-amylase and α-glucosidase is closely related to their structural properties [34]. Experimental results revealed that FPs showed a higher capacity of glycosidase activity inhibition (α-amylase inhibition rate: 47.22%, 0.11 mg AE/mg; α-glucosidase inhibition rate: 82.85%, 21.53 μg AE/g) than BPs (α-amylase activity inhibited by 32.88%, 0.01 mg AE/mg; α-glucosidase inhibition rate: 44.71%, 0.99 μg AE/g). The α-glucosidase inhibition rate of FPs extracted from the macroalgae residue was higher than that of the Nannochloropsis oculata extract (α-glucosidase inhibition rate: 80.42%) [35]. Studies have shown that FPs’ ability to inhibit glycosidase activity is stronger than that of BPs, which is consistent with the results of this experiment [36]. Notably, the disparity in glycosidase inhibitory activity between FPs and BPs was markedly greater than that observed for their antioxidant activities, and this phenomenon may arise from the difference in hypoglycemic activity of key differential constituents between these two polyphenols [8].

4. Conclusions

The optimized alkaline hydrolysis conditions led to a high BP extraction efficiency, demonstrating the effectiveness of this method for liberating cell-wall-bound bioactive compounds. A total of 23 polyphenols were identified, with 14 species for FPs and 9 species for BPs. This compositional divergence aligns with previous reports on algal polyphenol profiles, in which flavonoids dominated the free fraction. FPs exhibited greater abundance, and they contained one additional compound, potentially leading to a superior antioxidant ability and a higher glycosidase inhibition efficacy in FPs‌. There was a strong positive correlation between TPC, antioxidant activity, and glycosidase inhibition ability. This study verified macroalgae residues as a sustainable source of bioactive polyphenols, providing a preliminary understanding of their hypoglycemic activity and emphasizing their value as underutilized resources. Future research should focus on the quantitative determination of flavonoids and on elucidating the mechanism of the hypoglycemic effect and other biological activities.