Sample Collection and Materials Used

Discarded sole fish (P. erumei) samples were collected from the waterfront at Royapuram harbor (13°6′ 26 N 80° 17′ 43 E), Chennai, Tamil Nadu, India and validated by the Zoological Survey of India, Chennai. The muscle tissue was separated from the visceral mass, cleaned and stored at − 20 °C for further studies. O-phthalic aldehyde (OPA), dithiothreitol (DTT), Sodium dodecyl sulphate (SDS), Dimethyl sulfoxide (DMSO), LPS, Commercial proteases (Trypsin, Alcalase, Papain), 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and Sephadex G-25 beads were procured from Sigma-Aldrich (St. Louis, MO, USA). Amicon Ultra-15 centrifugation tubes were obtained from Merck Millipore Ltd., (Darmstadt, Germany). RAW264.7 cells were accessed from NCCS (Pune, India). Antibodies- Nucleolin (NUC) (14574S), NFκB-p65 (8242S), phosphor p-NFκB-p-p65 (3033S) from Cell Signaling Technology (CST; USA). Nitrocellulose membrane (NC) and Dulbecco's modified Eagle’s medium (DMEM) were bought from HiMedia Lab. Pvt. Ltd (Mumbai, India) and the remaining common chemicals used were of reagent grade.

Estimation of Protein and Amino Acid Analysis

The SM tissue samples were studied for the protein content. Moreover, quantification of amino acid was studied through reverse phase-high pressure liquid chromatography (RP-HPLC, 1100 series; Agilent Technologies Inc., Santa Clara, CA, USA). Moreover, acid hydrolysis was performed using 6 N HCl using crude SM samples with 24 h of stirring at 110 °C. After neutralizing the sample, 1 µl was injected into the Zorbax Rx-C 18 column (5 μm, 4.6 mmD × 150 mm)along with mobile phase A which was 7.35 mmol/L sodium acetate/triethylamine (500:0.12, v/v) and mobile phase B which was 7.35 mmol/L sodium acetate/methanol/acetonitrile (1:2:2, v/v/v). pH for both was adjusted to 7.2. Further, in order to separate different amino acids, the gradient of the flow was maintained as follows: 92% A and 8% B for initial 27.5 min (1 ml/min), 40% A and 60%B or next 4 min (1 ml/min). Further, 3 min wash with 100% B at 1 ml/min. The final eluant was equilibrated with 92% A and 8% B.

Protein Hydrolysate Preparation and Degree of Hydrolysis (DH)

Commercial proteases were used for the preparation of SM hydrolysates under physiological pH and optimum temperature conditions (Diniz and Martin 1997). Briefly, crude samples were dissolved in 0.1 M sodium phosphate buffer and the pH was maintained to 8, 8.2, and 7 for enzyme Trypsin, Alcalase, and Papain respectively. The enzymes were added to each sample based on the enzyme activity. For Trypsin the activity was 2000U/mg, Alcalase was 5000U/mg, and Papain was 6000U/mg. The temperature was maintained to 25 °C, 55 °C, and 70 °C uniformly throughout the experiment for Trypsin, Alcalase, and Papain treatments respectively. The samples were obtained at 3-h intervals upon uniform stirring up to 12 h. Furthermore, the inactivation of enzymes was done at 110 °C followed by centrifugation with a tabletop centrifuge (Allegra X-15R, Beckman Coulter, Calif., USA), and supernatants were stored at − 20 °C. The measurement of the peptide bond cleavage was calculated by DH using OPA reagent (6 mM OPA + 5.7 mM DTT + 0.1 M Borax and 2% SDS). Briefly, 3 ml of OPA reagent was added to 400 μl of the sample and incubated for 20 min at 27 °C while the absorbance was read at 340 nm through Cary 60 UV–vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). To calculate the total amino acid present in the protein hydrolysates, acid hydrolysis was performed using 6N HCl.

Estimation of Anti-inflammatory Activities

Protein denaturation inhibition (PDI) assay was performed using the method described by Chandra et al. (2012) with slight modifications. Briefly, a reaction mixture was prepared containing 2 ml hydrolysate (2 mg/ml), 0.2 ml egg albumin, and 2.8 ml of 0.1 M phosphate buffered saline (PBS). The mixture was mixed homogenously and incubated for 15 min following which it was heated at 70 °C and density was recorded at 660 nm. Diclofenac sodium and MilliQ water was used as positive control (PC) and negative control (NC) respectively.

Erythrocyte membrane protection (EMP) assay was performed according to the method of Balde et al. (2021). Briefly, a fresh human blood sample was collected and mixed with equal amount of Alsever solution. The mixture was centrifuged and the pellet was resuspended in isosaline solution (0.85% NaCl; pH 7.2) forming a suspension. To the hydrolysate sample; PBS buffer, erythrocyte cell suspension, and hyposaline solution (0.36% NaCl; pH 7.2) were added, incubated, and centrifuged at 3000 rpm. The absorbance for supernatant was further recorded at 560 nm and protection % was estimated.

Purification of Active Hydrolysates by UF and GFC

The enzyme hydrolysate which showed the highest percentage inhibition and protection were used for partial separation through UF (Rajapakse et al. 2005). Majorly, membrane centrifugal filter units with MWCO range of 30, 10 and 3 kDa were pre-rinsed with 0.1N NaOH following which the solubilized hydrolysates were added and centrifuged further. The fractions obtained (> 30, 30-10, 10-3, < 3 kDa) were lyophilized and further analyzed for protein content (%), yield (%) as well as anti-inflammatory assays.

The fractions showing the highest activity were further purified using a gel filtration column (2.5 × 75 cm) containing Sephadex G-25 beads which were pre-equilibrated using MilliQ water. The fractions were eluted at a flow of 1.2 ml/min using GE AKTA Prime plus (FPLC equipment, GE Healthcare) and the volume collected for each fraction was recorded at 280 nm to obtain chromatogram. Similarly, the peaks were lyophilized and tested for protein content (%), yield (%) as well as anti-inflammatory assays.

Sequence and Molecular Weight Determination

The active peak was collected and the sequence, as well as molecular mass, was examined using LC/MS–MS (Shimadzu, Kyoto, Japan).Positive ion mode was used to acquire the mass spectra where the mass spectrophotometer was coupled with OTOF detector (Bruker, Bremen, Germany). The fractions were dissolved in 0.1% formic acid and 1 µl of the sample was injected into the Shiseido polar C18 column (5 μm, 4.6 mmD × 150 mm) at room temperature and the flow rate was maintained at 0.4 ml/min flow rate. The mobile phase used here contained solvent A and Solvent B in 1:1 ratio which were prepared as 50% of 0.1% formic acid in water and HPLC grade acetonitrile respectively. The peptide sequence was determined by data analysis OTOF version 4.1, Bruker, Daltonics and the peptide sequence was confirmed using Biotools sequence editor, Version 3.2 (Bruker, Daltonics) software.

Functional Properties (Solubility, EAI, ESI, FC, and FS) of Purified Peptide

The solubility of the SM-derived peptide was determined. Briefly, peptide was dissolved in ultra-pure water and the solution was set at a physiological pH range of 2, 4, 6, 8, 10. Furthermore, post-centrifugation process, the supernatant was isolated and estimated for protein content to analyze the amount of peptide solubilized in the solvent. The emulsifying properties (EAI and ESI) of the SM peptide was estimated based on the method of Kristinsson and Rasco (2000). Briefly, the peptide was dissolved in corn oil at a fixed volume and homogenized. pH was adjusted for the suspensions to a range of 2, 4, 6, 8, 10 and centrifugated at 20,000 rpm at 37 °C. Further, the emulsion was collected at 0th min and 10th min which was dissolved in 0.1% SDS and record absorbance at 500 nm. Similarly, SM peptide was estimated for foaming properties (FC and FS). Herein, lyophilized peptide was dissolved in MilliQ water and pH range (2 to 10) was maintained. Initial as well as final volume was estimated upon vigorous homogenization to calculate FC and FS (Jeon et al. 1999).

Cell Culture and Cell Viability Assay

RAW264.7 cells were cultured in DMEM, enhanced with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin complex (Sigma-Aldrich, St. Louis, MO). The culture was seeded and incubated at 37 °C in a 5% CO2 incubator. The cell viability upon addition of peptide was estimated by MTT assay performed according to the method of Tang et al. (2011) with few modifications. Cells with a density of 1 × 104 cells/well were plated into a 96-well microplate. The cells were treated with SM peptide concentrations ranging from 10 to 100 μM for 24 h. To the wells, 20 µL MTT was added after 1× PBS wash and incubated in dark for 4 h. Further, 100 µl DMSO was added and absorbance was analysed at 570 nm. Triton-X 100 treated cells and untreated cells were tested asNC and PCrespectively.

Production of NO in Peptide Treated Macrophage Cells

Accumulation of nitrite in the culture medium was estimated by Griess reaction as mentioned previously by Hwang et al. (2019). The cells were seeded in the 96-well plate with a seeding density of 1 × 104 cells/well overnight and then stimulated with LPS for 24 h followed by the addition of peptide concentrations in the well. After the treatment, an equal volume of culture supernatant was mixed with Griess reagent and incubated for 10–15 min, and the absorbance was noted at 548 nm. Diclofenac sodium was used as standard control drug while untreated cells and LPS treated cells are used are PC and NC respectively.

Western Blot Analysis

The SDS-PAGE western blot analysis was performed using the method of Park et al. (2016). Briefly, the preheated nuclear extract samples for peptide treatment upon LPS stimulation were mixed with gel loading buffer and loaded into the wells of 10% polyacrylamide gel and the gel was run at 200 V and 360 mA. LPS stimulated and untreated cell nuclear extract was used as PC and NC respectively. Furthermore, the gel was transferred onto a NC membrane and blotting was performed. The membrane was blocked by 3% bovine serum albumin and washed further with PBS-Tween-20 (PBST). Also, specific primary antibody was added and incubated for 4 h. Further, membrane was washed with PBST and added with secondary antibody for 2 h. ECL kit (Bio-Rad, Hercules, CA, USA) at an enzyme/substrate ratio of 1:1 was used to analyse the protein bands on ChemiDoc XRS + blot analyzer as described and the intensity of the band was studied using ImageJ software.

Molecular Docking Analysis

For the docking analysis, 3D structures of peptide ligand (MTQML) was obtained through ChemDraw 19.0 (www.cambridgesoft.com) and receptor proteins iNOS (PDB ID: 1NSI), as well as NFκB-p65 (PDB ID: 1NFI), were retrieved from Protein Data Bank (http://www.pdb.org). The ligands and receptors were prepared for docking by MGL Tools 1.5.6 and the active site for inhibitors was predicted.To dock the peptide ligand against iNOS receptor protein, a grid box was prepared with centre coordinate as xyz: 11.644, 63.188, 15.995 respectively and 0.375 Å space. Similarly, docking against p65 receptor was performed around centre coordinates xyz: 3.159, 47.723, 17.303 respectivelyand 0.375 Å space. Further, the docking was executed through AutoDock Vina 1.0 at an energy range and exhaustiveness of 1.5 and 20 respectively (Maldonado-Rojas and Olivero-Verbel 2012). Gradient optimization algorithm was used for the molecular docking pose and the results are calculated over 10 runs each generating 9 configurations.Further, the lowest binding energy docking pose was selected and binding interactions were visualized through Discovery Studio visualizer 2020 software. The 2D models were studied for the types of interactions taking place between the peptide and the receptor protein residues.

Statistical Analysis

Statistical analysis was performed for all the assays through One-way ANOVA with SPSS 16.0 software (Chicago, IL, USA). All the experiments were performed in triplicates and the values were represented in the mean ± standard deviation format.