1. Introduction
Seaweeds, which are photosynthesizing macroalgae residing in the sea, comprise a diverse range of over 10,000 species identified globally [
1]. Owing to their nutrient-dense profiles, they are abundant in essential amino acids, proteins, polysaccharides, vitamins, and minerals. Moreover, they contain an abundance of bioactive substances such as fucoidans, galactans, phloroglucinol, and phlorotannins, which exhibit antioxidant, anti-inflammatory, anticancer, and anticoagulant activities [
2,
3,
4]. Notably, seaweeds are predominantly consumed in Asian countries such as Korea, Japan, and China [
5] and have applications across various domains, including diets, drugs, feeds, fertilizers, cosmetics, and biofuels [
6,
7,
8].
Seaweeds are categorized as brown (Phaeophyceae), red (Rhodophyceae), or green (Chlorophyceae) based on their pigmentation [
9]. Red seaweeds, primarily phycoerythrin and phycocyanin, inhabit both freshwater and seawater and prefer deeper and cooler seawater than their brown and green counterparts [
10]. Red seaweeds have been utilized as natural food dyes [
11], in paper development using red seaweed pulp [
12], and in eco-friendly packaging films [
13]. Notable red seaweeds include the genera
Porphyra,
Gelidium, and
Gracilaria, with
Gracilaria classified into 200 species, 10 of which have been reported in Korea, including
Gracilaria verrucosa (GV) [
14]. GVs are recognized as a food source for marine organisms such as abalone and crustaceans and are utilized as a primary source of agarose and bioethanol [
15,
16]. It is also consumed raw or added to salads in South Korea and used in salads and jelly in France, Malaysia, Vietnam, and Indonesia [
16].
Various edible seaweeds, such as
Sargassum fusiforme,
Undaria pinnatifida,
Saccharina japonica,
Ulva prolifera,
Porphyra tenera, and GV, are globally recognized [
17]. In 2020, these seaweeds were cultured to approximately 35 million tons globally, with
Gracilaria species contributing approximately 14.8% (5.18 million tons) of the total seaweed production, showing a consistent increase in production since 2020 [
18]. GV, which thrives in all coastal areas of Korea, has been studied for its carpospore release induction [
19], effects of growth conditions (light intensity and temperature) on early growth [
20], phylogeny [
14], and mineral content [
21]. Although previous studies have investigated its antiproliferative [
22], skin improvement [
23], antioxidant [
24], and antimicrobial [
25] activities, studies exploring its various biological activities in different extraction solvents are scarce. Consequently, this study investigated the antioxidant, antiaging, and antifungal activities of GV extracts in different extraction solvents (ethanol, methanol, and water), with the findings intended to serve as foundational data for the development of functional products using GV extracts.
2. Materials and Methods
2.1. Seaweed
Gracilaria verrucosa was obtained from Wando, Republic of Korea, in April 2020. The GV samples were washed three times to remove salinity and desiccated at 65 °C for 3 days. The desiccating GV materials were ground using a plant grinder (Hanil HMF-3970TG, Seoul, Republic of Korea) to facilitate the permeation of the extraction solvent during the extraction process. The samples were stored at −20 °C prior to extraction and finally lyophilized at −70 °C in a vacuum (SFDSF12, Samwoneng Eng, Busan, Republic of Korea) for 2 h.
2.2. Preparation of GV Extracts
For the GV water extract (GVW), GV samples were added to 50-fold distilled water (1:50
w/
v), soaked at 24 °C for 60 min, and extracted at 80 °C for 3 h. For the GV ethanol (GVE) and methanol extracts (GVM), GV samples were mixed with 10-fold 95% ethanol and 95% methanol (1:10,
w/
v) and extracted at 24 °C and 130 rpm for 1 d, respectively. The GV extracts were filtered through Whatman Grade 2 filters (Cytiva, Marlborough, MA, USA). The GVW, GVE, and GVM filtrates were concentrated using a rotary evaporator (Hei-VAP Core, Heidoiph-Ins., Schwabach, Germany) at 65, 37, and 37 °C, respectively. All the evaporation residues of the extracts were stored at −20 °C and then combined with extraction solvents prior to experimentation. The GV extract yield was calculated as follows:
where W
a is the extract weight and W
b is the freeze-dried GV sample weight.
2.3. The Total Polyphenol Content (TPC) Measurement
TPC was determined using a modified Folin–Denis method [
26]. The Folin–Denis reagent (Fluka, Switzerland) was mixed with 10 μg/μL GV extract and 10% sodium carbonate (Sigma-Aldrich Co., St. Louis, MO, USA) at an equal ratio, and the total reactant was incubated at 24 °C for 40 min. The absorbance of each reactant was measured at 760 nm, and a standard calibration graph was constructed using tannic acid.
2.4. The Total Flavonoid Content (TFC) Measurement
TFC was evaluated by modifying the method described by Nieva et al. [
27]. First, 10 μg/μL GV extract (5 μL) was mixed with 1 M potassium acetate (5 μL) and 10% Al(NO
3)
3 (5 μL), followed by extraction solvents (235 μL). The total reactant was incubated at 24 °C for 40 min. The absorbance of each reactant was measured at 415 nm, and a standard calibration graph was constructed using quercetin (QC), from which the total flavonoid content of the GV extract was calculated.
2.5. Antioxidant Activity Measurement
2.5.1. 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) Activity Measurement
The DPPH activity was assessed by modifying the Blois [
28] method to measure the reducing power of the GV samples. DPPH was dissolved in methanol, 50 μM of the dissolved DPPH (190 μL) was added to GV extracts (10 μL) at various concentrations, and the reaction was maintained at 24 °C for 15 min. The synthetic antioxidant butylhydroxytoluene (BHT) was prepared as a positive control group, and the DPPH assay was performed by measuring the absorbance of each sample at 490 nm, and the DPPH activity was calculated using the following formula [
28]. The concentration of the GV extract that scavenged 50% of the DPPH radicals is represented by its half-maximal inhibitory concentration (IC
50) value.
where A
sample is the absorbance of the reactant with the GV extract, and A
control is the absorbance of the reactant without the GV extract.
2.5.2. 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) Activity Measurement
To measure ABTS radical scavenging of GV extracts, 250 μL of 2.45 mM potassium peroxodisulfate mixed with 7 mM 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt was reacted under dark conditions at 24 °C for 15 h. Next, the ABTS solution was adjusted with distilled water until the optical density reached 0.6 at 734 nm [
29]. ABTS solution (180 μL) was added to the GV extract (20 μL) and kept at 24 °C for 4 min. The ABTS assay of all samples was performed by measuring the absorbance at 734 nm and calculating the absorbance using the susequesnt formula. The concentration of the GV extract that scavenged 50% of the ABTS radicals was represented as the IC
50 value.
2.5.3. Ferric Reducing Antioxidant Power (FRAP) Value Measurement
The FRAP reaction mixture was prepared from 10 mM tripyridyltriazine, 20 mM FeCl
3, and 0.1 M sodium acetate (pH 3.6) at a ratio of 1:1:10 (
v/
v/
v). The FRAP solution (150 μL) was mixed with GV extract (10 μL) and incubated at 37 °C for 20 min. Ascorbate was prepared as a positive sample, and the FRAP assay of all extracts was performed by measuring the absorbance at 593 nm and calculating using the formula [
30]. The GV extract concentration is represented as the half-maximal effective concentration of 50% of the FRAP capacity value.
2.6. In Vitro Antioxidant Enzyme Activity Measurement
The GV extract was mixed with a mixture (1:4, w/v) containing 50 mM KH2PO4 (pH 7.0), 1% polyvidone-40, and 1% triton X-100. The total mixture was incubated at 4 °C for 15 min. The centrifugation of the total mixture was carried out for 20 min (14,000 rpm; 4 °C). The supernatant was collected and quantified using a protein quantification kit (PCA kit, Thermo Fisher Co., Waltham, MA, USA), and antioxidant enzyme activity was measured.
The superoxide dismutase (SOD) reaction mixture was prepared with 50 mM KH
2PO
4 (pH 7.0), 3 mM xanthine, 3 mM EDTA, and 0.75 mM NBT. The GV extract (20 μL) was added to the SOD reaction mixture (200 μL) and maintained at 24 °C for 10 min. Then, the previous reactant and 0.4 unit/mL xanthine oxidase were mixed; the absorbance value of the final reactant was measured at 560 nm [
31]. The SOD activity of the samples was determined as follows:
Catalase (CAT) activity was determined by measuring the reduction in hydrogen peroxide (H
2O
2) according to the method described by Aebi [
32]. The GV extract (20 μL) was added to 50 mM KH
2PO
4 (pH 7.0) and a 10 mM H
2O
2 solution (200 μL). The absorbance of the final reactants was measured at 240 nm for 2 min. CAT activity in the samples was determined using the following formula:
where 43.6 is the H
2O
2 extinction coefficient at 240 nm.
Ascorbate peroxidase (APX) activity was measured according to the method described by Nacano and Asada [
33]. The ascorbate peroxidase (APX) mixture was prepared from 0.05 M KH
2PO
4 buffer (pH 7, 0.5 mM ascorbic acid (0.1 mM H
2O
2, and 0.1 mM) and EDTA. The GV extract (20 μL) was added to the APX mixture (200 μL) and reacted at 37 °C for 5 min. The absorbance of the reaction mixture was measured at 290 nm [
33]. The APX activities of the samples were calculated using the following formula:
where 2.8 is the extinction coefficient of ascorbate at 290 nm.
2.7. In Vitro Antiaging Activity Measurement
2.7.1. In Vitro Collagenase Inhibition Assay
A mixture of 1 μg/μL GV extract (50 μL) and 0.1 M Tris-HCl, pH 7.5, and 4 mM CaCl
2 solution (12.5 μL) was added to 0.5 μg/μL collagenase (75 μL). The mixture was reacted at 37 °C for 15 min and then added to 5% citric acid (0.25 mL) to stop the reaction. Finally, the mixture was added to ethyl acetate (750 μL), and the supernatant formed was dispensed into each well of a 96-well plate. The absorbance of the reaction mixture was measured at 320 nm. The collagenase inhibitory activity of the samples was determined as follows:
where A
se+ is the absorbance of the sample group added to the enzyme, A
se− is the absorbance of the sample group without the enzyme, A
ce+ is the absorbance of each extraction solvent added to the enzyme, and A
ce− is the absorbance of each extraction solvent without the enzyme.
2.7.2. In Vitro Elastase Inhibition Assay
The elastase inhibition assay of GV extracts was performed according to the method described by Cannell et al. [
34]. A mixture of 1 μg/μL GV extract (50 μL) was mixed with 0.5 μg/μL N-Succinyl-Ala-Ala-Ala-4-nitroanilide dissolved in 50 mM Tris-HCl, pH 8.6 (50 μL) and reacted at 25 °C for 10 min. After the reaction, the absorbance was measured at 410 nm, and then the reactant was added to 0.3 unit/mL elastase. The final mixture was kept at 25 °C for 10 min, and the absorbance was measured at 405 nm. The elastase inhibition assay was calculated as follows:
2.8. In Vitro Tyrosinase Inhibition Assay
The tyrosinase inhibitory activity of the GV extracts was measured according to the method described by Yagi et al. [
35], with modifications. First, 1 μg/μL GV extract (20 μL) was mixed with 1.5 mM 3-(3,4-Dihydroxyphenyl)-L-alanine dissolved in 0.1 M KH
2PO
4 reagent, pH 6.8 (70 μL), followed by reaction at 37 °C for 10 min. After the reaction, the reaction mixture was added to 500 units/mL of mushroom tyrosinase (50 μL) and incubated at 37 °C for 10 min. The tyrosinase inhibition assay was performed by measuring the absorbance at 490 nm as follows:
2.9. Measurement of Phenolic Compound Content
The phenolic compounds in the GV extracts were quantified using a liquid chromatography–tandem mass spectrometer (LC–MS/MS) (AB SCIEX 4000 Q-Trap, Concord, ON, Canada; Shimadzu LC 20A System, Kyoto, Japan) operated in negative mode under the following conditions: turbo ion spray, MRM scan type, with a temperature of 400 °C and a spray voltage of 4500 V. High-performance liquid chromatography (HPLC)-grade standards of three phenolic compounds (4-hydroxybenzoic acid, naringenin, and naringin) and solvents (acetonitrile and methanol) from Sigma Co., Louis, MO, USA, were utilized for analysis.
Initially, 20 mg of GV extract was dissolved in 1 mL of each solvent and centrifuged at 4 °C at 3000 rpm for 10 min, respectively. The supernatant was then diluted 40-fold with distilled water. Solutions of phenolic compound standards were prepared at a concentration of 10 mg/mL in methanol. Subsequently, 10 μL of each diluted extract and standard solution were injected into a Gemini C18 column (3 μm, 50 mm × 2.0 mm) maintained at 40 °C. The mobile phase, comprised of 0.1% formic acid in water and acetonitrile with 0.1% formic acid, was delivered at a flow rate of 0.3 mL/min. Samples were kept at 15 °C in the autosampler throughout the experiment. The elution gradient was set as follows: starting at 20% B for 0.5 min, ramping to 80% B over 2.0 min, holding at 80% B for 2.5 min, returning to 20% B for 2.6 min, and continuing for a total run time of 6 min. A standard calibration curve for the three phenolic compounds was generated and used to determine the phenolic compound content of the GV extracts.
2.10. Analysis of Antibacterial Activity
Six foodborne bacteria were used to evaluate the antibacterial activity of the GV extracts. The three gram-positive bacteria were
Bacillus cereus (KCTC 3062),
Staphylococcus aureus (KCCM 11764), and
Listeria monocytogenes (KCCM 40307); the three gram-negative bacteria were
Escherichia coli (KCCM 11234),
Salmonella typhimurium (KCCM 40253), and
Vibrio parahaemolyticus (KCCM 11965). The bacteria were obtained from the Korean Collection for Type Cultures (KCTC, Jeongeup, Republic of Korea) and the Korean Culture Center of Microorganisms (KCCM, Seoul, Republic of Korea). The culture conditions of each bacterium are shown in
Table 1. All bacterial strains were subcultured three times prior to the experiment.
The antibacterial activity of the GV extract was measured as the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) [
36]. The GVE, GVM, and GVW extracts were diluted with 8% ethanol, 8% methanol, and distilled water at 85 μg/μL, respectively. The diluted samples (50 μL) were dispensed by a 2-fold dilution method in each well of three 96-well plates, respectively. Each strain was used at 10
7 CFU/mL. Next, two-fold dilutions of the GV extracts (50 μL) were mixed with each strain (50 μL) and incubated at 37 °C for 24 h. After incubation, the suspension (40 μL) was placed onto a nutrient agar plate and incubated at 37 °C for 16 h, and the bacterial growth level on the agar plate was observed to determine the MIC and MBC.
2.11. Statistical Analysis
Statistical analyses were conducted using a one-way analysis of variance (ANOVA) with IBM SPSS Statistics 27 (SPSS Inc., Chicago, IL, USA). Significant differences between the data were assessed using Duncan’s multiple range test (
p < 0.05). Principal Component Analysis (PCA) was employed to elucidate the distinctions and groupings among the biological activities of the GV extracts derived using various solvent extraction methods. Additionally, partial least squares regression analysis (variable importance in projection [VIP] score) and correlation analysis were utilized to identify pivotal variables that delineated each group and to comprehend the relationships between GV extracts and their biological activities, employing MetaboAnalyst. Variables with a VIP score greater than 1 were considered significant in influencing the grouping of GV extracts [
37,
38]. All analyses were performed in triplicate and are presented as the mean ± standard deviation.
4. Conclusions
This study investigated the effects of different extraction solvents (95% methanol, ethanol, and hot water) on the antioxidant, antiaging, and antibacterial activities of GV. The TPC and TFC were measured and ranged from 0.8 to 4.0 mg TA/g and 0.2 to 0.9 mg QC/g, respectively. The highest TPC and TFC were identified in the GVM and GVE (3.74 ± 0.14 mg TA/g and 0.86 ± 0.06 mg QC/g, respectively). Stronger antioxidant activities (DPPH, ABTS, FRAP, and antioxidant enzyme activity) were observed for GVM and GVE than for GVW. In addition, the antiaging activities (collagenase, elastase, and tyrosinase inhibition activities) were the highest in the GVM. Finally, the highest antibacterial activity against the six foodborne pathogens was observed for GVE. Overall, the GVE and GVM had the highest TPC and TFC, suggesting that the high antioxidant and antibacterial activities of the GVE and GVM were influenced by the TPC and TFC of the GVE and GVM. These findings suggest that the phenolic and flavonoid compositions of GVM and GVE contribute significantly to their bioactivities and have potential applications in the development of functional agents in diverse industries. However, these results may vary under specific test conditions, and the efficacy of these compounds in real-world applications may vary. Therefore, additional research is needed to confirm these results.