Study on the antioxidant and antiosteoporotic activities of the oyster peptides prepared by ultrasound-assisted enzymatic hydrolysis

2.1. Extraction of total oyster protein

Oysters were purchased from a local seafood market (Shenzhen, China), transported to the laboratory on ice within 1 h, and then manually shucked to obtain oyster meat. Total oyster protein was prepared according to the previously described method [10]. Briefly, fresh oyster meat was washed with deionized water three times and then mixed with PBS (0.01 mol/L, pH = 7.2) at a ratio of 1:3 (m:v). The mixture was homogenized at 9000g for 120 min in an ice bath. Following homogenization, the mixture was centrifuged at 10,000g for 20 min at 4 °C, and the supernatants were obtained. The extraction steps were repeated, and the resulting supernatants were collected. The total oyster protein extract was obtained by freeze-drying. The yield and protein content of total oyster protein extract were 16.37 ± 0.48 % and 69.41 ± 3.57 %, respectively.

2.2. The production of peptides by ultrasound-assisted enzymatic hydrolysis

The total oyster protein hydrolysate (TOPH) was prepared following the method described by Liu et al., [20]. The total oyster protein powder was mixed with deionized water at a ratio of 1:7 (m:v). The mixture was pre-treated using an ultrasound cell pulverizer (JY88-IIN, SCIENTZ, China) operating at 20 kHz, with both on-time and off-time set to 2 s and a total treatment duration of 20 min in an ice bath, the ultrasound power was set as 0 W, 50 W, 100 W, 150 W, 200 W, 250 W, 300 W, respectively. After different ultrasound treatments, the substrate was enzymatically digested with pepsin (Macklin, Shanghai, China) for 2 h, followed by trypsin (Macklin, China) for an additional 2 h. Both pepsin and trypsin were used at a concentration of 5,000 U/g protein and the digestion was performed at 37 °C. The enzymatic reaction was inactivated at 100 °C for 10 min. The supernatant was then collected by centrifugation at 10,000 g for 10 min (4 °C), followed by lyophilization and storage at − 80 °C for subsequent use.

2.3. Degree of hydrolysis

The degree of hydrolysis of TOPH was assessed by the o-phthalaldehyde (OPA) method as described by Pan et al., [21]. Briefly, 100 μL of the sample solution was combined with 2 mL of freshly prepared OPA reagent. The absorbance was then recorded at 340 nm using a spectrophotometer (Infinite 200Pro, TECAN, Austria).

2.4. Soluble peptide concentration

The concentration of soluble peptides in TOPH was determined according to the previously described method of Gao et al., [22]. Briefly, the TOPH samples were mixed with 10 % trichloroacetic acid (TCA) in a ratio of 1:1 (m:v), and centrifuged at 3,000 rpm for 10 min. Peptide content in the supernatant was then quantified using Lowry’s method [23].

2.5. Scavenge OH· ability

The OH· scavenging activity was measured following the method described by Liu et al., [24]. Briefly, 10 mg of the sample was dissolved in 1 mL of deionized water, then mixed with 1 mL of 8 mM FeSO₄ and 1 mL of 3 mM salicylic acid. After mixing, 250 µL of 20 mM H₂O₂ was added. The absorbance was subsequently recorded at 536 nm, and the scavenging activity was calculated using the following equation:

2.6. Scavenge DPPH· ability

The DPPH· scavenging activity was determined as described by de Carvalho Oliveira et al., [25]. 10 mg of the sample was dissolved in 1 mL of ethanol and then mixed with 1 mL of DPPH solution (0.1 mM in ethanol) as sample groups. A blank control was prepared with a mixture of 1 mL of ethanol and 1 mL of DPPH solution. Following a 30-minute incubation period in darkness, the absorbance was assessed at a wavelength of 517 nm. Results were calculated using the following equation:

2.7. Scavenge ABTS⁺· ability

The scavenging activity of ABTS⁺· was measured by a modified method based on Zhang et al., [26] following the instructions provided with an ABTS assay kit (Cat#: S0121, Beyotime, Shanghai, China). Briefly, a 10 mg/mL sample solution was mixed with the ABTS working solution and allowed to react for 10 min. The absorbance was subsequently recorded at 414 nm. A control group was prepared using a reaction mixture without the TOPH. Results were calculated using the following equation:

2.8. Ferric reducing antioxidant power (FRAP)

The FRAP levels were quantified using an antioxidant capacity assay kit following the FRAP procedure (Cat#: S0116, Beyotime, China). Briefly, 10 mg/mL sample solution was combined with the FRAP working solution, and the absorbance was assessed at 593 nm. The FRAP values were then determined based on the linear calibration curve and denoted as μmol FeSO₄ equivalents.

2.9. Measurement of protein structure

2.10. Cell experiments

2.11. Identification of peptide sequences

The identification of peptide sequences was performed by Bioprofile Biotechnology Co., Ltd. (Shanghai, China). Briefly, the hydrolysate samples were dissolved in 0.1 % formic acid, desalted using a desalting column (C18 StageTip, Empore™, USA), and then re-dissolved in 0.1 % formic acid for LC-MS/MS analysis. The samples were subjected to chromatographic separation using Nano LC-MS/MS (EASY-nLC 1200, Thermo Scientific, USA). For this, an aliquot of the hydrolysate was injected into a Trap Column and separated on a gradient column at a flow rate of 300 nL/min. The peptides underwent separation and examination using a Q-Exactive Plus mass spectrometer (Thermo Scientific, USA). Data analysis was performed with MaxQuant 2.4.14.0.

2.12. Molecular docking

Bone morphogenetic protein type-2 (BMP-2, PDB ID: 3BMP) was used as a macromolecular receptor, retrieved from the protein data bank (https://www.rcsb.org/). Molecular docking was then conducted with peptides as flexible ligands and the receptors as rigid structures. Autodocktools 1.5.7 was employed to predict the binding sites between the peptides and receptors. The grid box was centered on the structural center of BMP-2, with additional parameters detailed in Table S1. The binding affinities of the docked complexes were evaluated, and the most optimal conformations were selected for further analysis. Docking was performed using Autodockvina, and the results were visualized using Pymol and Discovery Studio 2019 Client.

2.13. Peptide synthesis

Peptides were synthesized by GenScript Biotechnology Co., Ltd. (Nanjing, China). The purity of the synthetic peptides was confirmed to exceed 95 % by HPLC and mass spectrometry analysis (Fig. S1 and Fig. S2).

2.14. Statistical analysis

Statistical analysis was performed using SPSS 22.0 software, with results replicated at least three times and expressed as mean ± SEM. The analysis included one-way ANOVA followed by Duncan's multiple-range test. The significance difference was considered as a P-value < 0.05.

3.1. Effects of ultrasound on the extraction of peptides

The results of ultrasound-assisted enzymatic hydrolysis on the extraction of peptides showed that the degree of hydrolysis (Fig. 1A) and soluble peptide content (Fig. 1B) increased with increasing ultrasonic power, reaching a maximum at 200 W and then slightly decreased beyond 200 W. Specifically, the degree of hydrolysis of TOPH increased from 12.94 % at 0 W to 22.61 % at 200 W (Fig. 1A), and the content of soluble peptides rose from 22.53 g/100 g dw at 0 W to 32.09 g/100 g dw at 200 W (Fig. 1B). Likewise, it was found that the best extraction rate was achieved at 200 W when extracting Spirulina platensis peptide using ultrasonic pretreatment [27]. In this regard, previous studies have indicated that ultrasound treatment destabilizes protein structures and exposes additional sites for enzymatic hydrolysis, thereby enhancing enzymatic activity and improving peptide extraction [28], [29]. Furthermore, the cavitation effect of ultrasound may effectively disrupt protein cross-linking and aggregation, thereby reducing protein stability and enhancing solubility; however, prolonged exposure to high-power ultrasound may induce protein aggregation [30]. As ultrasound energy increases, surrounding bubble clusters may shield smaller bubbles near the solute, diminishing the impact of cavitation [14]. Together, these findings align with previous studies, suggesting that appropriate ultrasound treatment significantly enhanced protein hydrolysis and soluble peptide release.

3.2. Effects of ultrasound on the antioxidant activity of TOPH

Ultrasound power not only influences extraction efficiency but also affects the activity of protein hydrolysates [31]. To assess the effects of ultrasound on the antioxidant capacity of TOPH, we evaluated its ability to scavenge OH·, DPPH·, ABTS⁺·, and determined its FRAP value. As illustrated in Fig. 1C, the capacity of TOPH to scavenge OH· progressively increased with increasing ultrasound power, peaking at 200 W (39.86 %) and slightly decreasing at power levels above this threshold. Similarly, ultrasound treatment significantly increased the capacity of TOPH to scavenge DPPH· compared to the untreated group (0 W), reaching 66.84 % at 200 W (Fig. 1D). In the ABTS⁺· scavenging assay, the ability of TOPH to scavenge ABTS⁺· increased from 36.17 % at 0 W to a maximum of 55.20 % at 200 W (Fig. 1E). In the FRAP assay, ultrasound treatment significantly elevated the FRAP value compared to the untreated group, reaching a maximum of 0.88 μM Fe²⁺/mg at 200 W (Fig. 1F). These results align with those of ultrasound-assisted enzymatic hydrolysis on peptide extraction, indicating that ultrasound treatment enhanced the antioxidant activity of oyster hydrolysates, with optimal results achieved at 200 W. In line with this, Magalhães et al., [32] reported that the antioxidant capacity of protein hydrolysates pretreated by ultrasound-assisted enzymatic hydrolysis is typically proportional to the content of soluble peptides. Furthermore, moderate ultrasound is beneficial for the exposure of hydrophobic groups that contribute to the enhancement of antioxidant activity, whereas excessive ultrasound leads to protein aggregation and hinders the release of hydrophobic groups, thereby decreasing the antioxidant activity [14], [33]. These findings suggest that appropriate ultrasound treatment improved the antioxidant capacity of TOPH.

3.3. Effects of ultrasound on protein structure of TOPH

The UV–Vis absorption spectra in the range of 260–280 nm revealed that the peak intensity of TOPH subjected to ultrasound treatment was significantly higher than that of the samples without ultrasound treatment (Fig. 2A). The characteristic absorption at 260–280 nm may be attributed to hydrophobic aromatic amino acids, such as tyrosine, phenylalanine, and tryptophan [34]. Similar to the findings of Jin et al., [35] in the ultrasound pretreatment of corn gluten meal, appropriate ultrasound might promote cavitation and mechanical effects that can alter the spatial conformation of soluble proteins, leading to the exposure of hydrophobic amino acids. Yang et al., [33] showed that ultrasound may cause protein molecules to unfold due to intermolecular interactions, thereby exposing buried hydrophobic groups and increasing UV–Vis absorption intensity.

The circular dichroism results showed that, with increasing ultrasound power, there was a shift from the rigid α-helix structure to more flexible β-sheet and random coil structures (Fig. 2B). This shift is likely attributed to the cavitation effects and mechanical shear forces produced by ultrasound, which disrupt the protein structure, possibly unfolding the protein molecules and leading to a decrease in α-helix [36], [37]. In line with this, the α-helix portion of rice protein unfolded and transformed into β-sheet structures after being exposed to ultrasound [34]. Likewise, another study demonstrated that pretreatment of fibrin with ultrasound caused a reduction in α-helix levels and an increase in β-sheet levels [38]. It has been reported that a reduced α-helix content may expose peptide active sites more readily to free radicals, enhancing their scavenging activity [39]. Jiang et al., [40] reported that the decrease in α-helix content may be closely associated with the observed enhancement in antioxidant capacity.

The molecular weight distribution of protein hydrolysates is closely related to its functional properties [14]. As illustrated in Fig. 2C, the molecular weight distribution of TOPH was categorized into five ranges: < 500 Da, 500–1,000 Da, 1,000–3,500 Da, 3,500–8,000 Da, and > 8,000 Da. As ultrasound treatment power increased, the content of small molecular weight peptides (< 3,500 Da) increased, indicating a significant alteration in the molecular weight distribution of active peptides due to ultrasound treatment. The increased polypeptide content below 3,500 Da might be attributed to the higher degree of hydrolysis during ultrasound-assisted enzymatic hydrolysis [41]. These results were in agreement with a previous study, indicating an increase in the production of lower molecular weight fractions in sweet potato protein hydrolysates after ultrasound treatment [42]. Additionally, the increased low molecular weight fractions were associated with enhanced antioxidant activity [4].

Based on the above results, TOPH exhibited the best antioxidant activity when the ultrasound power was set at 200 W. Therefore, in the follow-up study, TOPH at 200 W (TOPH-200) was used to further investigate the antioxidant and antiosteoporotic activities of TOPH in osteoblastic MC3T3-E1 cells.

3.4. Effects of TOPH on H₂O₂-induced oxidative stress in MC3T3-E1 cells

Oxidative stress-induced osteoblast dysfunction plays a crucial role in the development of osteoporosis [43]. To investigate whether TOPH-200 can mitigate oxidative damage in osteoblasts, an H₂O₂-induced oxidative stress model in MC3T3-E1 cells was established. As shown in Fig. 3A, TOPH-200 was not toxic to MC3T3-E1 cells at concentrations of 0–500 μg/mL after incubation for 24 h. Given that TOPH-200 dose-dependently promoted osteoblastic cell proliferation in the dose range of 0–125 μg/mL, moderate concentrations of TOPH-200 at 5, 25, and 125 μg/mL were employed for further studies. As shown in Fig. 3B, the cell viability in the H₂O₂-treated model group decreased to 50.78 % compared with the blank control group. However, TOPH-200 treatment dose-dependently restored the cell viability, with survival rates increasing to 55.15 %, 61.87 %, and 65.72 % after 5, 25, and 125 μg/mL of TOPH-200 treatment, respectively. In addition, TOPH-200 dose-dependently decreased H₂O₂-induced intracellular ROS levels in MC3T3-E1 cells (Fig. 3C-D). The fluorescence intensity of ROS decreased by 12.85 % (5 μg/mL), 32.27 % (25 μg/mL), and 45.35 % (125 μg/mL), respectively, compared with the model group (Fig. 3D). The maintenance of redox balance within cells is greatly influenced by mitochondrial membrane potential (MMP), with decreased MMP levels serving as a primary indicator of cellular health [44]. Similar to the ROS results, TOPH-200 treatment effectively reversed the H₂O₂-induced decrease in MMP levels, which increased by 12.94 %, 58.59 %, and 76.70 %, respectively, after TOPH-200 treatment at 5, 25, and 125 μg/mL compared with the model group (Fig. 3E-F). Together, these results indicate that TOPH-200 effectively alleviated the H₂O₂-induced oxidative stress and mitochondrial damage in osteoblastic MC3T3-E1 cells.

3.5. Effects of TOPH on H₂O₂-induced osteogenic dysfunction in MC3T3-E1 cells

ALP is a fundamental component of the bone matrix and serves as an early indicator of osteoblast function and differentiation [1]. As shown in Fig. 4A-B, H₂O₂ treatment significantly reduced the ALP-stained area of MC3T3-E1 cells and decreased ALP viability levels, indicating impaired osteoblast differentiation. However, TOPH-200 effectively ameliorated the H₂O₂-induced decrease in ALP levels in a dose-dependent manner. TOPH-200 at 5, 25, and 125 μg/mL increased ALP activity by 10.39 %, 19.75 %, and 36.43 %, respectively, compared to the model group (Fig. 4A-B). ARS staining is commonly used to observe calcium nodules in osteoblasts by forming a deep red complex with calcium ions, serving as an essential indicator for quantitative mineralization analysis and osteogenic activity assessment [1]. As shown in Fig. 4C-D, H₂O₂ treatment significantly decreased the level of mineralized nodules in MC3T3-E1 cells. However, TOPH-200 dose-dependently increased the formation of calcium nodules compared with the H₂O₂-treated model group, showing a significant enhancement in mineralization at 125 μg/mL TOPH-200 (1.38-fold compared to the model group). These results indicate that TOPH-200 effectively ameliorated H₂O₂-induced osteogenic dysfunction in MC3T3-E1 cells, suggesting its potential antiosteoporotic activity.

3.6. Identification of the peptide sequences

TOPH-200 was subsequently used for the peptide sequence identification by nano-LC-MS/MS analysis. It has been reported that bioactive peptides typically consist of 2 to 20 amino acid residues [45], [46], [47]. As shown in Fig. 5, the number of peptides of different lengths in TOPH-200, where 248 peptide sequences were identified with high confidence (ALC% = 99 %), and most of the peptide sequences were composed of 9 to 18 amino acid residues, accounting for 81 % of the total 201 sequences.

PeptideRanker (https://distilldeep.ucd.ie/PeptideRanker/) is a tool employed to predict the bioactivity of peptides based on scores, with higher scores indicating greater bioactivity, and thresholds greater than 0.5 considered biologically active [14]. As shown in Fig. 5 and Table S2, a total of 26 peptide sequences (PeptideRanker score > 0.5) were predicted to be biologically active, representing 10.5 % of the total identified sequences. Moreover, 11 of the 26 peptides had a phenylalanine C-terminus, which has been suggested to directly transfer electrons to stabilize ROS, thereby exhibiting better antioxidant activity [48].

3.7. Molecular docking

The bone morphogenetic protein (BMP) pathway plays a crucial role in controlling osteoblastic differentiation and maturation at various stages. Among BMPs, BMP-2 has been extensively studied and frequently used for virtual analysis of the osteogenic activities of peptide ligands [49], [50]. To further screen peptides with antiosteoporotic activities, peptide sequences with predicted bioactivity scores > 0.8 were subjected to molecular docking with the BMP-2 protein (PDB ID: 3BMP). As shown in Table S3, the binding energies of four peptides with BMP-2 ranged from −6.1 to −5.8 kcal/mol, with DSQLAPFRF exhibiting the highest binding affinity to BMP-2 (−6.2 kcal/mol), followed by HFNPRL (−6.1 kcal/mol). In the docking study, a negative binding energy suggests that the binding occurs naturally, and it is considered more favorable to have lower values for the energy of the protein–ligand complex [5]. Therefore, the binding modes of DSQLAPFRF and HFNPRL with BMP-2 were further analyzed.

As shown in Fig. 6A1 and Fig. 6B1, both DSQLAPFRF and HFNPRL were located in the active pocket of BMP-2 and interacted with key amino acid residues through non-covalent interactions. Hydrogen bonds are acknowledged as among the most potent intermolecular forces [5]. Specifically, DSQLAPFRF formed hydrogen bonds with Tyr-38, Leu-84, Asp-105, and Met-106 of BMP-2, while HFNPRL interacted with BMP-2 through hydrogen bonds with Arg-9, Cys-43, Gly-45, Asn-68, Lys-76, and Cys-79 (Fig. 6A1 and Fig. 6B1). In this regard, a recent study highlighted that Cys-43 and Asp-105 are potential activation sites associated with BMP-2 signaling pathways related to osteogenesis [50]. The activation of BMP-2 has been demonstrated to upregulate the expression of osteoblast-specific genes, induce osteoblast differentiation and bone extracellular matrix synthesis and secretion, and promote their calcification and mineralisation [51]. These results suggest that HFNPRL and DSQLAPFRF may activate the osteogenic signaling pathway by interacting with Cys-43 and Asp-105 residues of BMP-2, respectively.

In addition to hydrogen bonds, other types of ligand-receptor interactions also play an important role in stabilizing protein–ligand complexes [4], [14]. Besides, the biological activity of protein peptides is closely related to the structural characteristics of the peptides, such as sequence and charge [4]. For DSQLAPFRF, its N-terminal amino group interacted with Asp-105 and Met-106 through N–H···O interactions, while Leu-84 provided the N-terminal amino group for the N–H···O interactions with DSQLAPFRF (Fig. 6A2). Additionally, carbon-hydrogen interactions between DSQLAPFRF and Met-106, as well as π-alkyl and π-σ interactions between the phenyl ring of phenylalanine and other residues, were observed (Fig. 6A2). For HFNPRL, the N-terminal amino group interacted with Cys-43, Asn-68, and Cys-79 through N–H···O interactions, while the guanidine N-terminal amino group of Arg-9 and the N-terminal amino group of Lys-76 contributed to the N–H···O interactions with HFNPRL (Fig. 6B2). Furthermore, interactions involving the imidazole ring of histidine and the phenyl ring of phenylalanine with other residues, including π-anion, π-cation, and π-σ interactions, were noted (Fig. 6B2). All these interactions might enhance the stability of both peptides at the BMP-2 binding site [4].

3.8. Verification of antioxidant and antiosteoporotic effects of synthesized peptides in MC3T3-E1 cells

To further investigate the antioxidant and antiosteoporotic activities of DSQLAPFRF (DS9) and HFNPRL (HF6), the effects of synthesized peptides on osteoblast proliferation, differentiation, and mineralization in the H₂O₂-induced oxidative damage model of MC3T3-E1 cells were evaluated. The cell viability results showed that both DS9 and HF6 were non-toxic to MC3T3-E1 cells at concentrations ranging from 0 to 50 μM after incubation for 24 h and promoted cell proliferation in a dose-dependent manner (Fig. 7A). Thus, peptides at 12.5, 25, and 50 μM were selected for subsequent experiments. In H₂O₂-induced oxidatively damaged MC3T3-E1 cells, both DS9 and HF6 effectively reversed H₂O₂-induced cell damage, with DS9 (50 μM) exhibiting the highest cell survival rate (80.48 %), followed by HF6 (50 μM; 76.02 %) (Fig. 7B). Additionally, both DS9 and HF6 dose-dependently enhanced the ALP staining area and cellular ALP activity in H₂O₂-treated MC3T3-E1 cells (Fig. 7C-D). Compared with the model group, pretreatment with DS9 (50 μM) and HF6 (50 μM) increased ALP activity by 0.46-fold and 0.38-fold, respectively (Fig. 7D). Furthermore, ARS staining results indicated that both DS9 and HF6 increased the formation of calcium nodules in H₂O₂-treated MC3T3-E1 cells. Quantitative analysis of ARS staining revealed that the absorbance of DS9 (50 μM) and HF6 (50 μM) groups increased by 1.03-fold and 0.64-fold compared with the model group, respectively (Fig. 7E-F). Taken together, these results indicate that DS9 and HF6 exhibit promising antiosteoporotic properties and effectively alleviate oxidative stress-induced impairments of osteoblast differentiation and mineralization.