Optimization of Phenolic Compounds in Subcritical Water Extracts from Ecklonia stolonifera by Response Surface Methodology

Abstract

Ecklonia stolonifera is a type of seaweed that belongs to the kelp family Seaweedaceae. Several studies have demonstrated its bioactive compounds. To extract these compounds, critical water hydrolysis was used, which is a green extraction technique. The reaction temperature of hydrolysis ranged from 120℃ to 220℃, and it played a crucial role in releasing active compounds such as sugars, phenols, flavonoids, and antioxidants. The results of temperature specific extraction indicate that the optimal temperature condition is 195℃. Therefore, we set the center temperature of the response surface methodology (RSM) at 195℃, and varied the temperature between 160℃, 195℃, and 230℃, the time between 20min, 40min, and 60min, and the ratio between 10 g/mL, 20 g/mL, and 30 g/mL. The optimal conditions for extraction temperature, time, and ratio were found to be 198.50°C, 36.21 min, and 12.23 g/mL. The individual phenolic compounds in the extract were identified using high-performance liquid chromatography (HPLC). The predominant phenolic compounds found were 4-hydroxybenzoic acid, chlorogenic acid, rutin, gallic acid, and 4-hydroxy-3,5-dimethoxycinnamic acid. E. stolonifera subcritical water extract exhibited high antioxidant activity in various assays, including ABTS+, DPPH, and FRAP. Under optimal conditions, the ABTS+ value was 70.94 ± 0.15 mg trolox equivalent/g dried sample, the DPPH value was 60.03 ± 0.35 mg trolox equivalent/g dried sample, and the FRAP value was 29.45 ± 0.39 mg trolox equivalent/g dried sample. These values were higher than those of the ethanol extract. Additionally, the physicochemical properties of E. stolonifera were analyzed, including TPC, TFC, TSC, and RSC. The TPC value for the optimal condition extract was 50.01 ± 0.12 mg phloroglucinol/g dried sample, and the TFC value was 43.58 ± 0.01 mg quercetin equivalent/g dried sample, which were approximately three times higher than the total phenolic content of the ethanol extract. The TSC and RSC values were higher in the ethanol extract, with 55.06 ± 1.51 mg glucose/g dried sample and 19.22 ± 0.23 mg glucose/g dried sample, respectively. The structural characterization (XRD, FT-IR) was also compared with the raw powder. The XRD analysis indicated high levels of KCl in the raw powder, while NaCl was predominantly observed in OCE-ES and ethanol extracts. The FT-IR analysis showed the presence of O-H, C-H, -C≡C-, O-C-O, and C-O groups in all three extracts. In the evaluation of antihypertensive activity, the positive control of 1% Captopril showed an activity of ≤99.22% ± 0.01%, while OCE-ES showed a higher activity of ≤95.87% ± 0.01% at a concentration of only 0.5%. Regarding anti-diabetic activity, the positive control of 1% acarbose showed ≤96.68% ± 0.07% activity, while OCE-ES showed ≤69.29% ± 1.83% activity and the ethanol extract showed ≤92.67% ± 1.13% activity. To test for anti-inflammatory activity, a positive control of 1% Diclofenac sodium was used and showed an activity of ≤66.80% ± 0.84%. OCE-ES showed an activity of ≤68.55% ± 0.98%, while the ethanol extract showed an activity of ≤41.62% ± 0.66%. This study utilized the subcritical water extraction method to determine the bioactivity of E. stolonifera extracts and confirmed their potential as a functional food material. Furthermore, these extracts can be applied to various industrial field.