Changes in the Biological Activities of Gracilaria verrucosa Extracted Using Different Extraction Solvents

Abstract

This study investigated the antioxidant, antiaging, and antibacterial properties of Gracilaria verrucosa (GV) based on 95% methanol (GVM), ethanol (GVE), and hot water (GVW) extractions. Antioxidant activity assays revealed the total polyphenol and flavonoid contents were highest in GVM and GVE. The 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) activities were highest in GVE and GVM. Furthermore, GVE exhibited the highest ferric-reducing antioxidant power (FRAP) value. In comparison, superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) activities were highest in GVM. Collectively, GVE and GVM had stronger antioxidant activities than GVW. Additionally, collagenase, elastase, and tyrosinase inhibition assays showed that GVM exhibited the strongest anti-wrinkle and skin-whitening activities. Liquid chromatography–tandem mass spectrometer (LC–MS/MS) revealed that GVW had the highest 4-hydroxy benzoic acid content, whereas GVE had the highest naringenin and naringin contents. Additionally, GVE exhibited the strongest antimicrobial activity against six foodborne bacteria, with minimum inhibitory and bactericidal concentrations of 0.06–0.3 and 0.1–0.5 μg/μL. Correlation analysis of the GV extracts indicated a strong positive relationship between TPC and ABTS, SOD, and CAT activities (r = 0.760–0.982, p = 0–0.018). Overall, GVE and GVM can be applied to the development of functional agents across diverse industries.

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:

$$\mathrm{Yield} (\%)=\frac{{\mathrm{W}}_{\mathrm{a}}}{{\mathrm{W}}_{\mathrm{b}}}\times 100$$

(1)

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(NO₃)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 (IC₅₀) value.

$$\mathrm{DPPH} \mathrm{activity} (\%)=\frac{{\mathrm{A}}_{\mathrm{sample}}-{\mathrm{A}}_{\mathrm{control}}}{{\mathrm{A}}_{\mathrm{sample}}}\times 100$$

(2)

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 IC₅₀ value.

$$\mathrm{ABTS} \mathrm{activity} (\%)=\frac{{\mathrm{A}}_{\mathrm{sample}}-{\mathrm{A}}_{\mathrm{control}}}{{\mathrm{A}}_{\mathrm{sample}}}\times 100$$

(3)

2.5.3. Ferric Reducing Antioxidant Power (FRAP) Value Measurement

The FRAP reaction mixture was prepared from 10 mM tripyridyltriazine, 20 mM FeCl₃, 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.

$$\mathrm{FRAP} \mathrm{activity} (\%)=\frac{{\mathrm{A}}_{\mathrm{sample}}-{\mathrm{A}}_{\mathrm{control}}}{{\mathrm{A}}_{\mathrm{sample}}}\times 100$$

(4)

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 KH₂PO₄ (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:

$$\begin{array}{c}\mathrm{SOD} \mathrm{inhibition} \mathrm{rate} (\%)={(\mathrm{A}}_{\mathrm{sample}}-{\mathrm{A}}_{\mathrm{control}})/{\mathrm{A}}_{\mathrm{control}}\times 100\\ \mathrm{SOD}\hspace{0.17em}\mathrm{unit}=\mathrm{SOD}\hspace{0.17em}\mathrm{inhibition}\hspace{0.17em}\mathrm{rate}\hspace{0.17em}(\%)/50\hspace{0.17em}(\mathrm{SOD}\hspace{0.17em}1\hspace{0.17em}\mathrm{unit}=50\%\mathrm{inhibition}\hspace{0.17em}\mathrm{rate})\\ \mathrm{SOD}\hspace{0.17em}\mathrm{units}/\mathrm{mg}=\mathrm{SOD}\hspace{0.17em}\mathrm{unit}\times \mathrm{dilution}\hspace{0.17em}\mathrm{factor}/\mathrm{Protein}\hspace{0.17em}\mathrm{concentration}\hspace{0.17em}(\mathrm{mg}/\mathrm{mL})\end{array}$$

(5)

Catalase (CAT) activity was determined by measuring the reduction in hydrogen peroxide (H₂O₂) according to the method described by Aebi [32]. The GV extract (20 μL) was added to 50 mM KH₂PO₄ (pH 7.0) and a 10 mM H₂O₂ 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:

$$\mathrm{CAT} \mathrm{activity} (\mathrm{unit}/\mathrm{mg})={[(\mathrm{A}}_{\mathrm{sample}}-{\mathrm{A}}_{\mathrm{control}})/43.6]\times \mathrm{dilution}\hspace{0.17em}\mathrm{factor}/\mathrm{Protein}\hspace{0.17em}\mathrm{concentration}\hspace{0.17em}(\mathrm{m}\mathrm{g}/\mathrm{m}\mathrm{L})$$

(6)

where 43.6 is the H₂O₂ 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 KH₂PO₄ buffer (pH 7, 0.5 mM ascorbic acid (0.1 mM H₂O₂, 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:

$$\mathrm{APX} \mathrm{activity} (\mathrm{unit}/\mathrm{mg})={[(\mathrm{A}}_{\mathrm{sample}}-{\mathrm{A}}_{\mathrm{control}})/2.8]\times \mathrm{dilution}\hspace{0.17em}\mathrm{factor}/\mathrm{Protein}\hspace{0.17em}\mathrm{concentration}\hspace{0.17em}(\mathrm{m}\mathrm{g}/\mathrm{m}\mathrm{L})$$

(7)

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 CaCl₂ 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:

$$\mathrm{Collagenase} \mathrm{inhibitory} \mathrm{activity} (\%)=\frac{{\mathrm{A}}_{\mathrm{se}+}-{\mathrm{A}}_{\mathrm{se}-}}{{\mathrm{A}}_{\mathrm{ce}+}-{\mathrm{A}}_{\mathrm{ce}-}}\times 100$$

(8)

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:

$$\mathrm{Elastase} \mathrm{inhibitory} \mathrm{activity} (\%)=\frac{{\mathrm{A}}_{\mathrm{ce}+}-({\mathrm{A}}_{\mathrm{se}}-{\mathrm{A}}_{\mathrm{se}-})}{{\mathrm{A}}_{\mathrm{ce}+}}\times 100$$

(9)

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 KH₂PO₄ 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:

$$\mathrm{Tyrosinase} \mathrm{inhibitory} \mathrm{activity} (\%)=\frac{{\mathrm{A}}_{\mathrm{ce}+}-({\mathrm{A}}_{\mathrm{se}+}-{\mathrm{A}}_{\mathrm{se}-})}{{\mathrm{A}}_{\mathrm{ce}+}}\times 100$$

(10)

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. Culture conditions of the foodborne bacteria used for the evaluation of the antibacterial activity of the GV extracts.

Foodborne Bacteria		Cultivation Conditions
		Culture Medium	Culture Temperature
Gram-positive	Bacillus cereus	Nutrient broth	30 °C
	Staphylococcus aureus	Nutrient broth	37 °C
	Listeria monocytogenes	Brain heart infusion broth	37 °C
Gram-negative	Escherichia coli	Nutrient broth	37 °C
	Salmonella typhimurium	Nutrient broth	30 °C
	Vibrio parahaemolyticus	Nutrient broth	37 °C

. 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 10⁷ 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.

3. Results and Discussion

3.1. Extraction Yield of GV Extracts

As shown in

Table 2. Yield of Gracilaria verrucosa extracts.

Extracts	GVE	GVM	GVW
Yield (%)	3.3 ± 0.3 b	1.3 ± 0.2 b	43.4 ± 1.7 a

GVE, G. verrucosa ethanol extract; GVM, G. verrucosa methanol extract; GVW, G. verrucosa water extract. Different letters (a–b) indicate significant differences (p < 0.05).

, the extraction yield of the GV extracts ranged from 3 to 44%, with the highest yield observed in the GVW at 43.4 ± 1.7%. Agar, a polysaccharide matrix located within the cell walls of red algae, is sourced from Gracilaria species and extracted at high temperatures [39]. The yield and quality of agar are contingent on factors such as the species of red algae, their growth environment, and extraction conditions [40,41,42]. High temperatures facilitate the aqueous extraction of agar, and methods such as alkaline treatment with 2% NaOH and 90–100 °C can alter agar’s structure, enhancing its yield [43]. Pereira et al. [44] observed the efficacy of high-temperature extraction of agar in Gracilaria vermiculophylla. Furthermore, another study showed that a 75% methanol extract of Gracilaria sp. yielded the highest extraction rate, suggesting that a combination of water and an organic solvent is beneficial for extracting compounds that are soluble in both media [45].

Therefore, the superior yield of GVW was likely attributable to the dissolution of soluble components at high temperatures during the extraction process. Additional studies are warranted to compare the extraction yields of GV using other solvents under consistent temperature conditions.

3.2. The Total Polyphenol and Flavonoid Content of GV Extracts

Phenolic compounds found in vegetables, fruits, grains, and seaweed consist of one or more aromatic rings and at least one hydroxyl (–OH) group [46]. These compounds are categorized into phenolic acids, flavonoids, stilbenes, and lignins and are known for their effective antioxidant activities, which are attributed to their free radical-scavenging ability [47,48]. The total polyphenol content of GV extracts using different extraction solvents varied from 0.8 to 4.0 mg TA/g, with the GVM exhibiting the highest content at 3.74 ± 0.14 mg TA/g (Figure 1a). Regarding the flavonoid contents, the GV extracts ranged from 0.2 to 0.9 mg QC/g, with the GVE and GVM displaying higher contents of 0.86 ± 0.01 and 0.64 ± 0.12 mg QC/g, respectively, than the GVW (Figure 1b). GVM and GVE had the highest total polyphenol and flavonoid contents, respectively, whereas GVW had the lowest.

Rusli et al. [49] observed that methanol extracts of GV, Sargassum sp., and Caulerpa recemosa had the highest polyphenol content, followed by ethanol and acetone extracts. Similarly, Farbin et al. [50] indicated that organic solvents are more efficacious than water for polyphenol extraction. The solubility of polyphenols is influenced by factors such as molecular size, the presence of hydroxyl groups, and hydrocarbon chain length [51]. These findings suggest that organic solvents such as methanol and ethanol are more effective than water for extracting polyphenol compounds from GV. However, comparisons of the yields of polyphenolic compounds based on extraction conditions such as temperature and solvent ratios, excluding extraction solvents, show considerable variability owing to factors such as the type of seaweed and the variability of standard reagents (such as gallic acid and tannic acid).

3.3. DPPH Activity of GV Extracts

DPPH is a purple free radical that can be used to analyze the antioxidant activity of a sample through color change [52]. The IC₅₀ of DPPH radical scavenging represents the extract concentration at which 50% of DPPH radical scavenging occurs; the lower the IC₅₀ value, the higher the DPPH activity. The DPPH activity of GV extracts ranged from IC₅₀ values of 69–182 μg/μL and increased in a concentration-dependent manner. The highest DPPH activity was observed in the GVE (IC₅₀ = 69.4 ± 0.4 μg/μL), followed by the GVM, while the GVW showed the lowest DPPH activity at IC₅₀ = 182 ± 17.8 μg/μL.

Sornalakshmi et al. [53] analyzed the DPPH activity of Gracilaria corticata using different solvent extracts, and their results showed that the ethanol and methanol extracts had higher DPPH activity than those of the other extracts (water, chloroform, and petroleum ether) and increased in a concentration-dependent manner. Similarly, Sobuja et al. [54] reported that methanol and ethanol extracts of Gracilaria tenuistipitata exhibit greater DPPH activity than water extracts. Antioxidants with DPPH activity also exhibit excellent electron-donating abilities [55]. In this study, GVM and GVE showed more effective DPPH activity than GVW, suggesting that the antioxidants in GVM and GVE stabilized free radicals more efficiently.

3.4. ABTS Activity of GV Extracts

ABTS activity is widely used to evaluate the antioxidant activity of samples, and the reduction rate of the ABTS radical caption was analyzed by monitoring absorbance [56]. ABTS is highly soluble in water and organic solvents, making it easy to measure the antioxidant activities of lipophilic and hydrophilic compounds [57]. The ABTS activity of the GV extracts in different extraction solvents was examined. The ABTS activity of GV extracts ranged from IC₅₀ = 53 to 277 μg/μL, with the GVE exhibiting the highest value (IC₅₀ = 53.5 ± 1.2 μg/μL) (

Table 3. DPPH, ABTS, and FRAP IC₅₀ values of Gracilaria verrucosa extracts.

IC50 = μg/μL	GVE	GVM	GVW	Ascorbic Acid
DPPH	69.4 ± 0.4 a	73.3 ± 0.9 a	182.0 ± 17.8 b	3.2 ± 0.0
ABTS	66.9 ± 2.0 a	53.5 ± 1.2 a	276.2 ± 20.7 b	0.1 ± 0.0
FRAP	56.1 ± 1.4 a	87.3 ± 0.9 b	171.6 ± 5.8 c	0.6 ± 0.0

GVE, G. verrucosa ethanol extract; GVM, G. verrucosa methanol extract; GVW, G. verrucosa water extract; IC₅₀, half-maximal inhibitory concentration. Different letters (a–c) indicate significant differences within the same row (p < 0.05).

). The ABTS activity of the GVW (276.2 ± 20.7 μg/μL) was 5.2- and 4.1-fold lower than that of the GVM and GVE, respectively. These results are similar to the previously analyzed DPPH activity of GV organic solvent extracts.

In another study, the antioxidant activities of Malaysian Gracilaria manilaensis extracts were as follows: ethanol > methanol > hot water, and the antioxidant activity was inversely proportional to the polarity of the extraction solvents [58]. Wang et al. [59] reported that phenolic compounds dissolve more easily in polar organic solvents than in water. Consequently, this study demonstrated the high polyphenol contents of GVE and GVM, which appeared to contribute to their high ABTS activity.

3.5. FRAP Activity of GV Extracts

FRAP activity was used to evaluate the capacity of the substances in a sample to reduce ferric ions to ferrous ions as electron donors, indicating that extracts with a reducing power can act as antioxidants [60]. The FRAP activity of GV extracts in different extraction solvents ranged from IC₅₀ = 56 to 172 μg/μL, and the highest FRAP activity was observed for the GVE (IC₅₀ = 56.1 ± 1.4 μg/μL) (Table 3). Furthermore, GVM exhibited higher activity than GVW. The lowest FRAP activity was observed in the GVW (IC₅₀ = 171.6 ± 5.8 μg/μL), which was 3-fold lower than that of the GVE.

Similar to the results reported by Chan et al. [61], the FRAP activity of the Malaysian red seaweed Gracilaria changii was highest, followed by ethyl acetate > ethanol > methanol > acetone extracts, with the water extract having the lowest FRAP activity. These results indicate that the type and content of phenolic compounds are influenced by the extraction solvents and methods used, which also affect the FRAP activity of G. changii. Furthermore, Neoh et al. [62] reported that the drying method of the red seaweed Kappaphycus alvarezii may affect its antioxidant activity. Therefore, further studies are required to investigate the antioxidant activity of GV under different conditions, extraction solvents, and extraction methods.

3.6. SOD, CAT, and APX Activities of GV Extracts

Plants remove toxic reactive oxygen species (ROS) generated by metabolic processes through reactions with antioxidant enzymes, including SOD, CAT, and APX [63]. SOD catalyzes the conversion of superoxide radicals (O₂^•–) into H₂O₂ and H₂O. Although H₂O₂ is toxic to cells, CAT and APX facilitate the conversion of H₂O₂ to H₂O and O₂ [64]. In this study, SOD, CAT, and APX activity assays were performed to estimate the antioxidant enzyme activities of the different GV extracts (Figure 2). The SOD activity of GV extracts ranged from 0.2 to 2.8 units/mg. Among the extracts, the GVM and GVE showed high SOD activities at 2.79 ± 0.40 and 2.26 ± 0.46 units/mg, respectively, followed by the GVW. Next, the CAT activity of GV extracts ranged from 0.0 to 0.6 units/mg, and the GVM showed 1.7- and 14-fold higher CAT activities than those of the GVE and GVW, respectively. Finally, the APX activity of the GV extracts ranged from 1.5 to 1.6 units/mg in APX activity. The GVM exhibited the highest APX activity, though not significantly higher than the other extracts.

Kurakake et al. [65] analyzed the SOD-like activity of the red Pyropia yezoensis and green Ulva sp. seaweed extracts, which showed high antioxidant activity when the extracts had a high polyphenol content. Kim et al. [66] reported that GVE has the highest polyphenol content and CAT activity, followed by Gloiopeltis furcata, Gracilaria vermiculophylla, and Chondracanthus tenellus ethanol extract. In this study, the highest polyphenol content in the GV extracts was observed in GVM, followed by GVE and GVW, and the SOD and CAT activities were similar. These results indicate that the polyphenol content of the extracts influences antioxidant enzyme activities and that GVM contains antioxidant substances related to the scavenging of superoxide and H₂O₂. In addition, El-Sheekh et al. [67] showed that treating tomatoes with seaweed extracts significantly increased the levels of antioxidant enzyme activities (SOD, CAT, and APX) and that the seaweed extracts enhanced resistance against tomato Fusarium wilt disease. Additionally, GVM appeared to be more effective in scavenging ROS than GVE and GVW because of the higher levels of SOD, CAT, and APX activity. These results indicate that GVM could potentially be used as a biological control agent. However, the antioxidant enzyme activity of seaweed extracts is affected by various conditions, such as light quality, temperature, and extraction conditions [68,69]. Therefore, measuring the antioxidant enzyme activity of GV under different light conditions, temperatures, harvest times, and drying methods is necessary in addition to the extraction method used in this study.

3.7. Analysis of Collagenase and Elastase Inhibition Activities of GV Extracts

Skin aging occurs naturally over time or is influenced by external factors such as ultraviolet (UV) radiation exposure [70]. Both exposure of the skin to UV radiation and the natural aging process produce free radicals, resulting in photooxidative damage and the induction of matrix metalloproteinases, including collagenase and elastase [71,72]. Collagen and elastin are the main structural proteins that constitute approximately 90% of the dermis and contribute to the elasticity and structural support of the skin [73]. However, collagenase and elastase degrade collagen and elastin, resulting in changes in the skin, including wrinkle formation, tough skin texture, and a lack of elasticity [74]. Thus, the inhibition of collagenase and elastase activities is the predominant factor in preventing skin aging [75]. Therefore, to confirm the anti-wrinkling ability of the GV extracts in this study, their collagenase and elastase inhibition activities were evaluated.

The collagenase inhibition activity of the GV extracts in different extraction solvents ranged from 0 to 17%, and the highest collagenase inhibition activity was observed for GVM, followed by GVE and GVW (

Table 4. Collagenase, elastase, and tyrosinase inhibition activities of Gracilaria verrucosa extracts.

Inhibition Activity (%)	GVE	GVM	GVW	Ascorbic Acid
Collagenase	10.1 ± 1.3 b	16.4 ± 1.0 a	0.9 ± 0.1 c	23.7 ± 1.1
Elastase	10.8 ± 0.4 c	21.2 ± 0.2 a	16.8 ± 0.1 b	52.2 ± 0.2
Tyrosinase	6.1 ± 0.5 c	37.9 ± 1.4 a	22.4 ± 1.5 b	50.2 ± 2.7

GVE, G. verrucosa ethanol extract; GVM, G. verrucosa methanol extract; GVW, G. verrucosa water extract. Different letters (a–c) indicate significant differences within the same row (p < 0.05).

). GVM showed a collagenase inhibitory activity of 16.4 ± 1.0%, which was 18-fold higher than that of the lowest GVW. The elastase inhibitory activity of the GV extracts ranged from 10 to 22%, and GVM showed the highest elastase inhibitory activity at 21.2 ± 0.2%, followed by GVW and GVE. Susano et al. [76] measured the collagenase inhibition activity of the brown seaweed Carpomitra costata according to solvent fractionation, and the ethyl acetate extract showed higher collagenase inhibition activity (IC₅₀ = 7.2 μg/mL) than that of other extracts. These results indicated that the high polyphenol content in the ethyl acetate extract affected its collagenase inhibitory activity. In this study, GVM had the highest inhibitory activity and the highest polyphenol content compared to the other extracts. These results indicate that the high polyphenol content in GVM may affect collagenase and elastase inhibition activities.

3.8. Analysis of Tyrosinase Inhibition Activity of GV Extracts

Melanin is a dark brown pigment that protects the skin from UV radiation [77]. However, excessive melanin accumulation can lead to hyperpigmentation, which manifests as melasma lesions and freckles [78]. Tyrosinase catalyzes the biosynthesis of melanin from melanocytes and is an effective agent for skin-whitening effects. Therefore, this study analyzed tyrosinase inhibition activity to investigate the skin-whitening effects of GV extracts (Table 4). The tyrosinase inhibitory activity of GV extracts was in the range of 6.0–38.0%, with the highest activity observed in GVM, followed by GVW and GVE. Although GVM showed significantly higher tyrosinase inhibitory activity (37.9 ± 1.4%) than GVW and GVE, it demonstrated lower activity than ascorbic acid (50.2 ± 2.7%), which was used as the positive control.

Cha et al. [79] examined the tyrosinase inhibition activity of 40 seaweeds extracted at 20 °C and 70 °C and reported high activity in aqueous extracts of red seaweed Schizymenia dubyi, brown seaweed Endarachne binghamiae, Ecklonia cava, and Sargassum siliqustrum. These results show a difference in tyrosinase inhibitory activity depending on both the extraction temperature and species. Paudel et al. [80] reported differences in the tyrosinase inhibitory activity of extracts depending on the substrate used (such as 3,4-dihydroxy-L-phenyl-alanine and L-tyrosine). Therefore, further studies are required to evaluate the tyrosinase inhibitory activity of GV extracts under different conditions, including different extraction methods and substrates.

3.9. Analysis of the Phenolic Compounds Content in GV Extracts

Phenolic acids comprise a carboxyl group and a hydroxyl group (-OH) on the benzene ring and are classified as hydroxybenzoic acid and hydroxycinnamic acid [81,82]. Among them, 4-hydroxy benzoic acid (4-HBA) is a phenolic compound found in diverse seaweeds [83,84] and has been used as an antioxidant and food preservative [85]. Naringenin and naringin are flavonoids mainly present in citrus and grapefruit; naringenin is an aglycone form of naringin [86]. Naringin is associated with the bitter taste of grapefruit and is used as a food additive [87]. Naringenin and naringin have antioxidant, anticancer, and anti-inflammatory activities [88,89,90]. Thus, the 4-HBA, naringenin, and naringin contents in the GV extracts were analyzed to develop functional products and increase their applicability using GV extracts (

Table 5. Three phenolic compound contents in Gracilaria verrucosa extracts.

Compound Contents (μg/g)	GVE	GVM	GVW
4-hydroxy benzoic acid	ND	ND	1.91 ± 0.01
Naringenin	1.33 ± 0.01	ND	ND
Naringin	666.88 ± 4.07	ND	ND

GVE, G. verrucosa ethanol extract; GVM, G. verrucosa methanol extract; GVW, G. verrucosa water extract; ND, not detected.

). The 4-HBA content in GVW was 1.91 ± 0.01 μg/g; however, 4-HBA was not found in the GVM and GVE. Meanwhile, the GVE showed naringenin and naringin contents of 1.33 ± 0.01 and 666.88 ± 4.07 μg/g, respectively; however, GVW and GVM did not contain these compounds.

Capillo et al. [91] reported that the 4-HBA contents of Gracilaria gracilis extracts were found in the methanol, ethanol, and acetone extracts (167.00, 124.93, and 59.36 mg/100 g, respectively). These results differed from the 4-HBA contents in the GVM and GVE in this study. Notably, bioactive components in seaweeds, including polyphenols, polysaccharides, and amino acids, are affected by storage and drying conditions [92]. Overall, various conditions, such as extraction methods, extraction solvents, sample storage, and drying methods, could have affected the phenolic compound content of the seaweeds.

3.10. Analysis of the Antibacterial Activity of GV Extracts

To investigate the antibacterial activity of the GV extracts against foodborne bacteria, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the GV extracts against the six bacteria were determined (

Table 6. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Gracilaria verrucosa extract against foodborne bacteria.

Foodborne Bacteria	GVE		GVM		GVW
	MIC (μg/μL)	MBC (μg/μL)	MIC (μg/μL)	MBC (μg/μL)	MIC (μg/μL)	MBC (μg/μL)
B. cereus	0.25	0.5	2.67	5.31	-	-
S. aureus	0.125	0.25	2.67	5.31	-	-
L. monocytogens	0.0625	0.125	1.33	2.67	-	-
E. coli	0.125	0.25	1.33	2.67	-	-
S. typhymurium	0.125	0.25	2.67	5.31	-	-
V. parahaemolyticus	0.125	0.25	1.33	2.67	-	-

GVE, G. verrucosa ethanol extract; GVM, G. verrucosa methanol extract; GVW, G. verrucosa water extract; -, not decorated.

). The MIC values for GV extracts ranged from 0 to 3 μg/μL against these bacteria. Notably, the GVE exhibited the highest MIC values (0.06–0.3 μg/μL) compared to the other extracts. The MIC values for the GVM ranged from 1 to 3 μg/μL. In contrast, GVW was not effective against foodborne bacteria, suggesting that it possessed the lowest antibacterial activity among the extracts. The GV extracts displayed MBC values ranging from 1 to 6.0 μg/μL against the six foodborne bacteria. The GVE had MBC values in the range of 0.1–0.5 μg/μL, showcasing the highest antibacterial activity, followed by the GVM and GVW. Specifically, the GVE and GVM demonstrated the most potent antibacterial activity against L. monocytogenes (MBC values = 0.125 and 2.67 μg/μL, respectively).

Polysaccharides, such as alginic acid, carrageenan, and laminarin, serve as protective agents against pathogens and promote the accumulation of proteins or compounds with antimicrobial properties [93]. Pérez et al. [94] reported that the yield of bioactive substances from seaweeds is influenced by the extraction solvent. Hydrophilic solvents, such as methanol, ethanol, and acetone, exhibit superior antimicrobial activity compared to hydrophobic solvents. Alshuniaber et al. [95] noted that the methanol fraction of Cyanobacteria Spirulina platensis showed high polyphenol content and antimicrobial activity against E. coli and S. aureus and that the polyphenols from the methanol fraction interacted with each other, resulting in strong antimicrobial activity of the methanol extract, even at low concentrations. Dayuti [96] explored the antibacterial activity of GV against E. coli and S. typhimurium based on the extraction solvent and administration time. Contrary to our findings, they found that a methanol:aqueous ratio of 75:25 and an extraction time of approximately 72 h yielded the most potent inhibitory activity against both bacteria.

Belhaoues et al. [97] reported that the ethyl acetate fraction of Anthemis praecox Link was observed to contain a high amount of phenolic compounds, especially naringin (68.7 µg/mg), which exhibited antibacterial activity against Staphylococcus aureus and Enterococcus faecalis. In our results, the GVE had the most abundant naringin content (666.88 µg/g) and displayed the lowest MIC and MBC values. This suggests that the high naringin content and interaction of its phenolic compounds contribute significantly to the antibacterial properties of GVE. Both GVE and GVM demonstrated antibacterial activities against foodborne bacteria. However, to employ them as antimicrobial agents, the functional compounds within GV extracts must be isolated, identified, and evaluated for safety.

3.11. Principal Component Analysis (PCA) of Biological Activities of GV Extracts

Principal component analysis (PCA) was performed to analyze the patterns of TPC, TFC, and antioxidant activity in the GV extracts. The principal component1 (PC1) and the principal component2 (PC2) explain 88.3 and 10.8% of the total variance, respectively (Figure 3a). PC1 was mainly related to TPC and ABTS activity and to the horizontal axis variance of the GV extracts. APX activity, GVW, and GVM were distributed in the positive direction of the PC2 axis, and FRAP activity and GVE were distributed in the negative direction of the PC2 axis. As shown in Figure 3b, the arrow exists in the opposite direction between GVW and TPC, TFC, and antioxidant activity, suggesting low antioxidant activity. GVM and GVE showed that the arrows existed in equal directions, indicating that GVM and GVE had high levels of TPC, TFC, and antioxidant activity.

Furthermore, the variable importance in the projection (VIP) score was analyzed to determine the variables affecting the separation of GV extract groups. The VIP scores for the ABTS, DPPH, and FRAP activities were 1.64, 1.40, and 1.32, respectively (Figure 4a). Among the biological activities, ABTS, DPPH, and FRAP activities were confirmed as factors that distinguished the GV extracts using different extraction solvents. The extract of the red seaweed G. changii, which contains polyphenol compounds and has excellent radical-scavenging activity, showed a strong positive correlation with TPC, TFC, and DPPH activity [62]. Similarly, GV extracts exhibited significantly high positive correlations (r = 0.760–0.982, p = 0–0.018) with TPC and SOD, CAT, and ABTS activity (Figure 4b). These results revealed that the high antioxidant activity of the GV extracts could be attributed to their total polyphenol content.

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.