The sample collection has been approved by the local authorities and the owner of the Sea grapes pond. Fresh Sea grapes (Caulerpa racemosa) were collected from the Sea grapes cultivation pond in Jepara Regency, Central Java Province, Indonesia (6°35'12.5"S latitude 110°38'36.0"E longitude). Botanical identification and authentication were confirmed in the Integrated Laboratory of the Faculty of Sciences and Technology (Herbarium Laboratory), UIN Sunan Kalijaga, Yogyakarta-55281, Indonesia, conducted by Dian Aruni Kumalawati, M.Sc and then followed by confirmation by biologist Prof. Dr. Trina Ekawati Tallei (Expert and Professor of Biology), and has complied with National Center for Biotechnology Information (NCBI) Taxonomy ID 76317 (Eukaryota/Viridiplantae/Chlorophyta/Ulvophyceae/Bryopsidales/Caulerpaceae/Caulerpa). Specimens were collected for future reference. Researchers (authors) state and confirm that all methods carried out in this study are in line or in accordance with relevant guidelines and regulations of in vitro and algae study.
2.1. Sea Grapes Extract Preparations
Sea grapes (C. racemosa) were thoroughly washed so that the dirt attached to the sea grapes becomes lost and clean. Washed Sea grapes were then twisted and dried in an oven (Memmert Incubator IN55) at a temperature of 60 °C for 3 x 24 hours. Sea grapes (whole-body) were dried, cut into small pieces, and then mashed with a blender to obtain Sea grapes simplicia powder (C. racemosa). Dried simplicia was mashed and then extracted using two methods: the hot and cold ways. Maceration represents the cold way, while Soxhlet extraction represents the way of heat.
2.1.1. Maceration Extraction Method
A total of 1,000 g of simplicia powder Sea grapes (C. racemosa) were put in a dark bottle, then 96% ethanol solvent (C₂H₅OH; Merck) as much as 2 L with a ratio of 1:2 between simplicia and solvent were mixed and soaked for 3 x 24 hours. Every 1 x 24 hours, the acquired filtrate was occasionally stirred, then filtered with Whatman 41 paper, and the residue was re-macerated with a new 96% ethanol solvent. The extracted sample was concentrated using a rotary evaporator (RV 8 IKA) under low pressure (100 millibars) for 90 minutes and re-evaporated in the oven (Memmert Incubator IN55) at a temperature of 40 °C so that a thick extract of Sea grapes were obtained. The extract was stored in the refrigerator at a temperature of 10 °C until used in research.
2.1.2. Soxhlet Extraction Method
Fifty grams of Sea grapes simplicia powder (C. racemosa) was wrapped in filter paper and inserted into a Soxhlet tube (thimble) on installed Soxhlet tools (PYREX® Soxhlet extractor). 96% ethanol solvent (C2H5OH; Merck) along with 250 ml of the solution were divided into two parts; 150 ml was inserted into the Soxhlet gourd (pumpkin round base), and 100 ml was inserted into the Soxhlet tube to moisten the simplicia. The ratio between simplicia and solvent was 1:5. The Soxhlet extraction process was carried out at a temperature of 70 – 80 °C, and the extraction was carried out for up to 3 repeat cycles. The extract was stored in the refrigerator at a temperature of 10 °C until used in research.
2.1.3. Hydrolysis Extraction Method by α-Chymotrypsin for Bioactive Peptide Measurements
C. racemosa simplicia was dissolved in a 1.0 mM phosphate buffer and then hydrolyzed with the α-chymotrypsin enzyme under its optimal conditions, referring to 20, which utilizes 1,200 U/mg enzyme activity, 37 °C temperature, at 8.0 pH with digestion time of 2 hours, substrate concentration of 20 mg/mL, 4.0 E/S (w/w) (%). The reaction was stopped by heating at 95 °C for 15 minutes, and the hydrolysate protein was centrifuged at 16,000 rpm for 10 minutes at 4 °C. The supernatant protein (PS) was lyophilized and stored at a temperature of -20 °C for further use in the analysis of the bioactive profiling of peptides.
2.2. Metabolomic Profiling Extract from Maceration and Soxhlet Methods
The untargeted metabolomics profiling test on Sea grapes extract samples (Maceration; Soxhlet Method) was carried out using the Liquid Chromatography High-Resolution Mass Spectrometry (LC-HRMS) method at the Laboratorium Sentral Ilmu Hayati (LSIH; ISO 9001:2008 and ISO 17025:2005; Central Laboratory of Life Sciences; Brawijaya University, Malang-65145, Indonesia) testing services, with the test number 041/LSIH-UB/LK/II/2022.
2.2.1. Analysis of the Maceration and Soxhlet samples by HPLC-ESI-HRMS/MS
Fifty (50) μl of extract samples (Maceration; Soxhlet Method) were diluted using 96% ethanol up to a final volume of 1,500 μl. The solutions were vortexed at 2,000 rpm for 2 minutes and then span-down at 6,000 rpm for 2 minutes. The supernatant was taken and then filtered using a 0.22μm syringe filter and injected into the vial. The sample in the vial was ready to be inserted into an autosampler and then injected into LC-HRMS (Liquid Chromatography High-Resolution Mass Spectrometry). LC-HRMS uses High-Performance Liquid Chromatography (HPLC) Thermo Scientific Dionex Ultimate 3000 RSLC nano with microflow meter. Solvents A and B consist of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. The analytical column uses Hypersil GOLD aQ 50 x 1 mm x 1.9 μ particle size with a flow rate of 40 uL/min, a flow gradient run time of 30 minutes, and a column oven with a temperature of 30 °C. High-Resolution Mass Spectrometer using Thermo Scientific Q Exactive with a full scan at 70,000 resolution, data-dependent MS/MS at 17,500 resolution, and run time of 30 minutes, with both positive and negative mode.
2.2.2. Processing Data Software
Annotated or detected compounds were automatically identified via mzCloud MS/MS Library (Thermo Scientific Q Exactive Software), which were performed by Midia Lestari Wahyu Handayani, S.TP, M.Sc., MP., Ph.D., a certified laboratory technician at the Sentral Ilmu Hayati Laboratorium (LSIH; ISO 9001:2008 and ISO 17025:2005; Central Laboratory of Life Sciences; Brawijaya University, Malang-65145, Indonesia).
2.3. Proteomics Assay of Sequence and Molecular Weight of Amino Acids
2.3.1. Ultrafiltration and Reversed-Phase HPLC (RP-HPLC)
The hydrolyzed protein (PS) of C. racemosa was divided into 3 peptide fractions based on molecular weight ((F1; < 3 kDa), (F2; 3–10 kDa), and (F3; > 10 kDa)) using ultrafiltration membrane of 10 kDa and 3 kDa. This approach referred to 20, which showed that peptide fraction of F1 (MW < 3 kDa) at 4 mg/mL had better antioxidant activity than F2 (3 – 10 kDa) dan F3 (MW > 3 kDa). Peptides with lower molecular weight are more active than those with a high molecular weight 20.
2.3.2. Separation of fraction F1 by RP-HPLC
Fraction 1 (F1; < 3 kDa) was purified using RP-HPLC. 40 mg of peptide fraction was dissolved into 1 mL 0.05% TFA (v/v) and then was filtered using micropores membrane (0.22 m) before being inserted into Agilent ZORBAX SB-C18 (5 m, 9.4 × 150 mm). A binary moving phase system was used in this study along with eluent A (0,1% TFA (v/v)) and moving phase B (ACN, 0.05% (v/v) TFA). The solution was eluted with a linear gradient of 0 – 40% moving phase B from 0 – 40 minutes dan 40% moving phase B from 40 – 55 minutes. All fractions were collected and lyophilized for further activity assays. The purification level of fractions with the highest activity was further analyzed using the Sunfire C18 column (5 m, 4,6 mm × 150 mm; Waters, USA). The column was eluted using a linear gradient of 0 – 20% moving phase B from 0 – 20 minutes and 20% moving phase B from 20 – 25 minutes, resulting in a purified fraction 1 (PF1).
2.3.3. Analysis of Amino Acid Sequence and MW
The ELWKTF (Glu-Leu-Trp-Lys-Thr-Phe) peptide was synthesized in the Laboratory of Biochemistry and Biomolecular Medicine, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia. A protein sequencer from Applied Biosystems 494 (ProciseTM 494 N-terminal sequencer; Applied Biosystems Inc, Foster City, CA, USA) was used to examine the amino acid sequence of the purified fraction (PF1) based on time-of-flight quadrupole mass spectrometer (MS/MS) paired with electrospray ionization (ESI) source to determines the molecular weight of the ELWKTF (Glu-Leu-Trp-Lys-Thr-Phe). Analysis of the sequence and molecular weight of ELWKTF was performed according to the method described by Zhang et al. (2019) 24. The results of research by Xiaoqian Zhang et al. 2019 showed good activity of ELWKTF (Glu-Leu-Trp-Lys-Thr-Phe) against radical scavenging activity (DPPH and ABTS) 20.
2.4. DPPH Antioxidant Radical Scavenging Activity Assay
The percentage (%) of the inhibition of 2,2-diphenyl-1-picrylhydrazyl (DPPH) was measured using a method referring to Kaur et al., (2021) 25 and Permatasari et al., (2022) 26, on all samples which include a macerated extract (EM), soxhlet extract (ES), and purified fraction 1 (PF1), while glutathione (GSH; 354102, Sigma-Aldrich) was used as a positive control. In the testing vial (at a concentration of 1, 2, 3, 4, 5 μg, an aliquot (100 μL) of samples and control was added, followed by a DPPH reagent addition (3 mL). The DPPH-extract combination that resulted was then left undisturbed (30 min; dark cycle). The samples were read at 517 nm absorbance with a UV-Vis Shimadzu 80 spectrophotometer. To ensure the validity of the data results, each sample was checked three times (n = 3). Inhibition of DPPH was expressed as a percentage and is determined according to the formula below:
A0 = Absorbance of blank; A1 = Absorbance of standard or sample.
The half-elimination ratio (EC50) was used to express the radical scavenging capacity of EM, ES, PF1, and GSH and defined as the concentration of a sample that caused a 50% decrease in the initial radical concentration.
2.5. ABTS Radical Scavenging Activity assay
For testing the 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) or diammonium salt radical cation (ABTS+; Sigma-Aldrich), the procedure follows the method introduced by Arnao et al. (2010) with some modifications 27. The stock solution includes 7 mM ABTS solution and 2.4 mM potassium persulfate solution. The working solution was prepared by mixing two stock solutions in equal quantities and letting them react for 14 hours at room temperature under dark conditions. The solution was then diluted by mixing 1 mL of ABTS solution with 60 mL of ethanol to set absorbance to 0.706 ± 0.01 units at 734 nm using a spectrophotometer (Thermo Scientific™ GENESYS™) is obtained. A fresh or new ABTS fresh/new solution was prepared for each test. Samples (at a concentration of 1, 2, 3, 4, and 5 μg) were allowed to react with 1 ml of ABTS solution, and the absorbance was taken at 734 nm after 7 minutes using a spectrophotometer. Treatment was carried out in the same way for all samples, including extract maceration (EM), extract-soxhletation (ES), purified fraction 1 (PF1), and Trolox (6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; Sigma-Aldrich) was used as a positive control. All determinations were performed in three replication (n = 3).
A0 = Absorbance of blank; A1 = Absorbance of standard or sample.
The half-elimination ratio (EC50) was used to express the radical scavenging capacity of EM, ES, PF1, and GSH and defined as the concentration of a sample that caused a 50% decrease in the initial radical concentration.
2.6. Cytotoxicity Evaluation using MTT Assay
Cell viability was assessed on the Human Caucasian skin fibroblast cell line (Normal cell; Bud-8) 28. The proliferation rate of the Bud-8 cell line after sample treatment was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. A mitochondrial dehydrogenase reduces MTT to a purple compound formazan that is insoluble in water, depending on the viability of the cell. Cells were preserved in Dulbecco's Modified Essential Medium (DMEM), which was supplemented with 10% fetal bovine serum (FBS) and 1x Penicillin-Streptomycin-Neomycin (PSN). One hundred microliters of cells (4 × 104 cells/mL) were seeded in a 96-well plate and incubated at 37°C, 5% carbon dioxide for 24 hours. After 24 hours of incubation, the cells were treated with 100 μL of 100, 200, 300, 400, and 500 μg/mL of samples (EM, ES, PF1). The plate was incubated at 37°C, with 5% CO2 for 24 and 48 hours. After incubation, the morphology of the cells was examined under a microscope. Twenty microliters of MTT (5 mg/mL) (Sigma) solution was added to each well plate. The plate was further incubated for 2 to 4 hours, and the medium was removed. Formazan crystals dissolved with 100 μL dimethyl sulfoxide (DMSO; Sigma). Absorbance was measured at 560 nm, and the percentage of cell viability and LC50 cells is calculated by:
Where A0 is absorbance control in cells given 1%, DMSO and A1 are cells' absorbance samples given the test sample.
Lethal concentration (LC50) is the lowest concentration of samples that inhibits 50% of cells. In general, a low LC50 value indicates high toxicity. Extracts with high LC50 are preferred for use due to their low toxicity effect on host cells 28.
2.7. Data Management and Analysis
Data from in vitro tests (DPPH antioxidants, ABTS antioxidants, and Cytotoxicity) were analyzed for significance or not between groups (EM, ES, PF1, or control) using two-way ANOVA CI 95% (0.05) with the MacBook version of GraphPad Prism 9.0.0 premium software. All data were presented in the form of average ± SEM. Graphic visualizations were presented using the MacBook version of GraphPad Prism 9.0.0 premium software. The graphical abstract was designed using the author's licensed BioRender Premium (Fahrul Nurkolis).