Dual Bioactivity of Angiotensin Converting Enzyme Inhibition and Antioxidant Novel Tripeptides from Sipunculus nudus L. and Their Related Mechanism Analysis for Antihypertention

The pathogenesis of hypertension is related to many factors including active angiotensin-I-converting enzyme (ACE) and excess reactive oxygen species (ROS), the combination of ACE inhibition and antioxidant activity in one compound might be useful for the treatment of hypertension. Sipunculus nudus L contain high protein content, which can be used as a valuable raw material to prepare bio-active peptides. In this study, two novel tripeptides, LPK and PRP, with dual bioactivity of ACE inhibition and antioxidant were purified and identified from the worm. The antioxidant activities of LPK and PRP are dose-dependent, including 2,2-diphenyl-1-picrylhydrazyl (DPPH), superoxide anion and hydroxyl radical scavenging activities. Moreover, the antioxidant activity of PRP was stronger than that of LPK. In addition, LPK and PRP also show higher ACE inhibitory activity, with IC50 of 6.54 and 0.42 mM, respectively. Molecular docking simulation showed that both the peptides could interact with the active ACE sites via hydrogen bonds, which can reduce the production of Ang II, thereby; LPK and PRP treatment could decrease Ang II-triggered superoxide production, and meanwhile, the peptides treatment could directly remove excess reactive oxygen species. The above-mentioned advantageous effects appeared to involve the stress of the RAS and scavenging ROS, both were benefit to treat hypertension. This research showed that LPK and PRP had potential to be developed as antihypertension agents, which might be a new strategy for high value utilization of Sipunculus nudus L as functional food material.


Introduction
Sipunculus nudus L., the Chinese name as Shachong, is high economic value seafood. Because of its delicious taste and nutritional benefits, it is very popular in Southeast Asia areas (Ge et al. 2018). It contains a lot of proteins, polysaccharides, and mineral salts; the proteins content of dry body was up to 85% (g/g dry body weight) (Cao et al. 2021). S. nudus is a type of Sipuncula phylum and widely distributed in subtidal zones or seabeds along the coasts from Western Pacific, Atlantic to Indian Ocean Dai 2011a, 2011b). Recent years, it is massively cultivated in Beibu Gulf, South China, as a top marine resource that Huan Liu and Xuaixuan Cai have equally contributed to this work.
3 Page 2 of 15 offers excellent and untapped bioactive ingredients. In addition, there has long been an extensive use of Shachong in traditional folk medicine to treat nocturia, tuberculosis, and regulate the functions of spleen and stomach . In recent years, the biological property of S. nudus has become an attracted research topic, including immunomodulatory, anti-hypoxic, anti-thrombus, anti-fatigue, anti-radiation and antioxidant, anti-inflammatory and antinociceptive activities Li et al. 2016;Su et al. 2016;Ge et al. 2018). These bioactivities were mainly related with the polysaccharides of S. nudus, little information is available on its protein and hydrolyzed peptide. In fact, numerous functional proteins and peptides contain in the worm. Based on our previous study about the peptides extraction and utilization of S. nudus (Qi et al. 2022), the antihypertensive activity of S. nudus peptides were further investigated.
The World Health Organization (WHO) reported that there were more than 1 billion people suffer from hypertension in the world (Oparil et al. 2018). The pathogenesis of hypertension is quite complex and it relates to many factors including active Angiotensin-I-Converting Enzyme (ACE) and excess Excess reactive oxygen species (ROS) (Asoodeh et al. 2014).
ACE is a zinc metallopeptidase with function to regulating blood pressure. ACE can convert inactivate angiotensin I to angiotensin II, the potent vasoconstrictor, in renin-angiotensin system (RAS) (Lassoued et al. 2015). Obviusly, the inhibitors of ACE are considered to be therapeutic agents of hypertension. Currently, several synthetic ACE inhibitors have been used as antihypertensive drugs in clinical prescriptions, such as captopril, alacepril, fosinopril, perindopril ,benazepril and others dicarboxylate derivatives (García et al. 2013;Luna-Vital et al. 2015). Unfortunately, these drugs could lead to harmful side-effects including kidney damage, vomiting, persistent dry cough, and so on (Antonios and MacGregor 1995). By contrast, natural ACE inhibitors isolated from plants or animals can be considered as safer alternatives (Abachi et al. 2019).
Excess ROS is produced in vasocular endothelial cells from oxidative stress resulting in an oxidation/antioxidation imbalance, which can lead to excessive free radicals including hydroxyl radicals, superoxide radicals, alkoxy radicals and peroxyl radicals (Bhuyan and Mugesh 2011;Kizhakekuttu and Widlansky 2010). Inevitably, the accumulation of free radicals leads to vascular damage, which is one inducement of high blood pressure. Vascular dysfunction is a major consequence of oxidative stress, as ROS damage vascular wall cells and result in dysfunctional vascular endothelial cells. Natural peptides are useful for the treatment of hypertension with both of ACE inhibition and antioxidant activity (Gaikwad et al. 2021).
Dual bioactivity of ACE inhibition and antioxidant in one product could be highly effective for controlling hypertension. There were some protein hydrolysates, such as hen egg white lysozyme, rapeseed, and fermented milk were reported to show both ACE inhibitory and antioxidant activities ( Rao et al. 2012;Yu et al. 2013;Moslehishad et al. 2013). However, these studies reported the two activities separately; how they jointly affect blood pressure and how they were related in the mechanism of antihypertension remained unclear.
In this work, we attempted to a dependable method to hydrolyze S. nudus proteins and optimize hydrolysis process by response surface methodology (RSM). Novel peptides were isolated from these hydrolysates and identified the amino acid sequences; their ACE inhibitory and antioxidant activity were investigated by Hippuryl-Histidyl-Leucine (HHL) substrate assay and free radical scavenging assay, respectively; mechanism analysis of ACE and ROS jointly influencing hypertension was performed with molecular docking and cell culture assay.

Materials
Fresh S. nudus, identified by Professor Si-dong Li (Guangdong Ocean University), was obtained from Bishuiwan Aquatic Products Company in Zhanjiang (N 21° 12′; E 110° 4′), China. The worm was washed, viscera was removed, homogenized, and stored at − 20 °C until use. The enzyme hydrolysis of S. nudus was based on our previous study (Qi et al. 2022).
(USA); all other reagents arose out of analytical grade.

Optimization of Hydrolysis Conditions
Response Surface Methodology (RSM) was used to optimize the enzyme hydrolysis parameters. A Box-Behnken design (BBD) was employed to evaluate the effects of three

Purification and Identification of S. nudus Peptides
The hydrolysate was ultrafiltrated into fractions using membranes with molecular weight cutoff (MWCO) 2.5 kDa, 5 kDa and 10 kDa at 4 °C. Each retentate was collected separately and named as SF-I (MW < 2.5 kDa), SF-II (MW 2.5-5 kDa), SF-III (MW5-10 kDa), SF-IV (MW > 10 kDa). The ACE inhibitory activity and radical scavenging activity of the peptide fragments were measured. The peptide fraction with the highest activity was then further separated by gel filtration chromatography (Task-gel G3000SW; Tosoh, Tokyo, Japan). The amino acids sequences of peptides were determined by liquid chromatography and tandem mass spectrometry (LC-MS/MS), and then the identified peptides were mapped through the internet databases BIOPEP-UWM, PepBank and EROP-Moscow.

Properties of Peptides
It is important for future research to understand the properties of the target peptides. A Swiss server (http:// www. swiss targe tpred iction. ch/) has been set up to predict the likely targets of bioactive molecules. The online database can be used to retrieve these parameters. The solubility of the peptides can be evaluated using the online Innovagen server (http:// www. innov agen. com/ prote omics-tools), and the bioactivity can be predicted using Peptideranker server(http:// biowa re. ucd. ie/).

ACE Inhibitory Activity Assay
The ACE inhibitory activity assay was performed following the method with slight modifications (Cushman and Cheung 1971;Wu et al. 2002;Zhao et al. 2019). Briefly, 100 µl sample with 10 µL ACE solution (0.1 U/mL) was pre-incubated at 37 °C for 10 min, and then incubated with 100 µl HHL (5 mmol/L) at 37 °C for 60 min, then added 150 mL glacial acetic acid to terminate the reaction. HPLC (1260 Infinity II, Agilent Technologies Co., Ltd.) was used to quantify the product hippuric acid (HA). The chromatographic conditions: Mobile phase was 10% acetonitrile with 90% of 0.5% (v/v) acetic acid aqueous solution, flow rate 1.0 mL/min and UV detection at 228 nm. IC 50 represents the peptide concentration that inhibits half of the ACE activity. ACE inhibitory rate was calculated by equation: Where HA inhibitor was the area of HA peak with inhibitor. HA control was the area of the HA peak without inhibitor.

Molecular Docking of ACE and Peptides
Molecular docking between peptide and ACE was performed by using Discovery Studio 3.5 software (DS 3.5, Accelrys, San Diego, CA, USA). The structure and minimum energy forms of target peptides were automatically calculated based on their amino acid sequences. The crystal structure of human tACE (PDB ID: 1O8A) was obtained from RCSB Protein Data (Tu et al. 2017). The 3D structure of target peptides was designed and energetically minimized by Discovery Studio 3.5. The binding site sphere was x: 42.232, y: 33.7204 and z: 44.9686. Finally, the pictures were generated using PyMOL and Discovery Studio 3.5 (Schiffrin et al. 2020).

DPPH Radical Scavenging Assay
The antioxidant capacity of S. nudus peptide was determined using the DPPH radical scavenging assay according to Shiqi Yang ). 2 mL sample was mixed with 1 mL solution of DPPH (200 mg/L) in ethanol, the mixture shaked at room temperature, stand in dark for 30 min; the absorbance of the sample was measured at 517 nm by a spectrophotometer (Biotek Epoch). Vitamin C was used as a positive control. DPPH free radical scavenging activity was calculated as follows.
where A 0 and A 1 represent the absorbance of the blank and test samples, respectively. IC 50 value was defined as the peptides concentration that could scavenge half of the hydroxyl radical.

Hydroxyl Radical (OH·) Scavenging Assay
The OH· scavenging capacity of peptide was measured as described method by Brands with minor modifications (Brands et al. 2019). Briefly, 1mL sample mixed with 2 mL phosphate buffer (0.2 M, pH 7.4), 2 mL orthophenanthroline (0.1 mM), 1 mL FeSO 4 solution (0.15 mM) and 1 mL H 2 O 2 . After 60 min incubation at 37 °C, the absorbance was read at 550 nm. The hydroxyl radicals scavenging activity was calculated as following equation: where A 0 represent the absorbance of the reaction solution without sample, A 1 represent the absorbance of the sample with water and A 2 represent the absorbance of the sample with H 2 O 2 .

Superoxide Anion (·O 2− ) Scavenging Assay
The superoxide radical scavenging assay was measured according to Yang (Yang et al. 2019). In brief, the reaction mixture contained 50 µL the sample, 150 µL nitroblue tetrazolium (43 mM), and 50 µL phenazine methosulphate (2.7 mM). After incubation in dark for 20 min at 37 °C, the OD value was recorded at 560 nm. The superoxide anion radical scavenging activity was calculated as following equation: Scavenging activity where A 0 is the absorbance of blank group (without sample) and A 1 is the absorbance of sample group.

Statistical Analysis
All experiments were implemented with At least 3 replicates, and the results were presented as X ± SD deviation. The experiment data were analyzed by ANOVA test and independent-sample T-test using GraphPad Prism 6.01. The p-value < 0.05 was considered statistically significant.

RSM Model Building and Statistical Analysis
The RSM was performed to investigate the impact of the independent variables on S. nudus hydrolysis. The experimental conditions and the response values of ACE inhibition are given in Table 2. The response values of ACE inhibition are from 36.70 to 65.50%, and the polynomial equation was as follows: The Design Expert 10.0.7 software was used to generate three-dimensional (3D) response surface graph and contour plot, which indicated their interactions about the hydrolysis parameters including temperature, time, typsin dosage, and their combined effect on ACE inhibition. The simultaneous effect of temperature and time on ACE inhibition showed in Fig. 1a, temperature and dosage (Fig. 1b), time and dosage (Fig. 1c) on ACE inhibition could be also obtained. The results obviously indicated, with the increase of the enzymatic reaction temperature, the inhibitory effect of ACE increased continuously. When the enzymatic hydrolysis temperature reached up to a certain value, the ACE inhibition reached the maximum value, and then showed a downward trend. This may be attributed to too high temperature leading to inactivation of the enzyme, which leaded to inadequate enzymatic hydrolysis. Similarly, the enzymatic hydrolysis time and typsin dosage were optimized. The results of the statistical tests were shown in Table 3. The F value for the regression model of ACE inhibition was 14.76 (p < 0.050) which indicated the model term was significant (Kim et al. 2020). The p-value was 0.39 (p > 0.050), which demonstrated the experimental data adapted to the model. Furthermore, the linear coefficients (X 1 , X 2 , X 3 ) and quadratic term coefficients (X 1 2 , X 2 2 , X 3 2 ), with p < 0.05, had insignificant effects. The determination coefficient (R 2 ) was considered as the percentage of the explained variation to the total variation (Tu et al. 2017). The R 2 value of the model was 0.9499, indicating that there was a high correlation between the predicted value and the measured value. The adj-R 2 and the Pred-R 2 value were 0.8856 and 0.7595, respectively, and both were reasonably consistent.
Based on the above results, the predicted optimal hydrolysis conditions were obtained as follow: enzymolysis temperature 52.5 °C, time 4.14 h, typsin dosage 0.33%, and the predicted response value of ACE inhibition was 61.79%. These values were consistent with the experimental values of ACE inhibition of 60.78 ± 6.50% at 52.5 °C for 4 h with 0.33% typsin dosage.

Fractions of S. nudus Hydrolysate by Ultrafiltration
Ultrafiltration is widely used to separate hydrolysates to obtain the target active fractions . In this study, the S. nudus hydrolysate was further ultrafiltrated to obtaine four fractions as SF-I, SF-II, SF-III, SF-IV. The fractions were lyophilized and weigh to calculate the yield, moreover, their ACE inhibitory and DPPH scavenging activity were determined, the results were given in Table 4. Y(ACE inhibition %) = 60.98 + 0.43 X 1 + 3.17 X 2 + 3.58 X 3 + 2.25 X 1 X 2 + 1.18 X 1 X 3 − 1.81 X 2 X 3 − 5.71 X 2 1 − 10.01 X 2 2 − 5.43 X 2 3 SF-I showed the highest ACE inhibitory and DPPH radical scavenging activity, compared with other fractions. These results were consistent with previous report that peptides with molecular weight below 3 kDa had higher biological activity (Mirzapour-Kouhdasht et al. 2021). Boschin et al. (Boschin et al. 2014) reported that small peptides with molecular weight less than 3 kDa possessed strong ACE inhibitory activity, the reason was that small peptide was easy to enter the ACE active site (Lin et al. 2017). Chi et al. (Chi et al. 2014) reported that lower molecular weight of peptides generally exhibited the greater free radical scavenging activities, which smaller molecular weight fractions interact more effectively with free radicals. To sum up, smaller molecular weight fractions performed higher ACE inhibitory and antioxidant activity.

Purification and Identification of Peptides
Because of its highest ACE inhibitory and DPPH radical scavenging activity, SF-I was selected for further purification through gel chromatograph and the result was shown in Fig. 2. The amino sequences of the isolated peptides were then identified by ESI-TOF-MS/MS. Two novel tripeptides were identified, and their sequences were Leu-Pro-Lys (LPK; m/z = 356.46 Da) and Pro-Arg-Pro (PRP; m/z = 368.43), the result was shown in Fig. 3. The amino acid sequence of Pro-Arg-Pro was found in Uniprot databases (https:// www. unipr ot. org/ unipr ot/). Both tripeptides had not previously been reported from S. nudus. The physicochemical properties of peptides presented in Table 5. The isoelectric points (PI) of LPK and PRP were 10.12 and 11.29, respectively. Both peptides have 1 net charge at PH = 7 and also shown good solubility in water. Compared with LPK, PRP has a higher prediction score of biological activity and thus may have better biological activity.
Among the structural features of ACE-inhibiting peptides, the most important is the length of the peptide. It has been confirmed by measuring many peptides that peptides containing 2-12 amino acids have better ACE-inhibiting activity, which was because oligopeptides were easily absorbed into the bloodstream to resulting good bioavailability (Aluko 2015;Hernández-Ledesma et al. 2011).

ACE Inhibition Activity of LPK and PRP
Both LPK and PRP showed significant ACE inhibitory activity, and the IC 50 values were 6.54 ± 0.910 and 0.42 ± 0.002 mM, respectively, the IC 50 value of captopril was 0.51 nM as a reference (Aydin et al. 2021). According to literature reports, peptides with more hydrophobic amino acids such as Ala, Phe, Gly, Leu, Ile, Pro and Val have better ACE inhibitory activity (Abdelhedi et al. 2018;Lin et al. 2017  In the identified tripeptides, the proportion of hydrophobic amino acids was high up to 66% in both LPK and PRP. Both LPK and PRP showed ACE inhibitory activity, PRP was significantly stronger than that of LPK, which might be related to the different amino acids contained in the C-terminal and/ or N-terminal of the peptides. The N-terminus of LPK and PRP both contained hydrophobic amino acids, which made the both tripeptides show good ACE inhibitory activity. The C-terminus of LPK was the amino acid Lys, while the C-terminus of PRP contains the hydrophobic amino acid Pro, it was well known that hydrophobic amino acid at the C-terminal could increase the binding ability of the peptide to ACE (Kheeree et al. 2020), thus the ACE inhibitory activity of PRP was stronger.

Molecular Docking of Peptide and ACE
The affinity of LPK and PRP for ACE was evaluated using the CDOCKER module and CHARMm's molecular dynamics (MD) method. CDOCKER was often used to study the interaction mechanism between ligands and receptors. -CDOCKER energy and -CDCKER interaction energy were the results of evaluating CDOCKER. The higher the energy scores, the stronger the ACE inhibitory activity was. The -CDOCKER energies and -CDCKER interaction energies of LPK and PRP were 45.9967, 50.4597 kcal/mol and 64.261, 69.995 kcal/mol, respectively. The energy scores of PRP were higher than that of LPK, indicating the affinity of PRP on ACE was stronger than that of LPK. The study found that there are three main active pockets S1 (Ala354, Glu384 and Tyr523), S2 (Gln281, His353, Lys511, His513 and Tyr520) and S10 (Glu162) in the ACE molecule. In addition, ACE also has a zinc ion (Zn 2+ ) active site, and it can bind to His383, His387 and Glu411. These active pockets and sites of ACE can bind to ACE inhibitors such as captopril (Natesh et al. 2003;Sturrock et al. 2004;Wang et al. 2017). It was shown in Fig. 4 (2D) that LPK and PRP bind to the active pocket of ACE mainly through electrostatic force and van der Waals force, and the tighter the polypeptide binds to the active site residue, the better the activity was, among which PRP has the best effect. The binding modes of LPK and PRP to ACE were shown in Fig. 5 (3D), and PRP forms eight hydrogen bonds with residues GLU162, GLN281, Ala354, GLU384, Tyr520 and Tyr523 of ACE. LPK only forms two hydrogen bonds with residue Tyr523 of ACE (Table 6).  LPK cannot form hydrogen bonds with many active site residues because of far distance. Hydrogen-bonding interactions played an important role in stabilizing the docking complex (Ishak et al. 2021), so LPK activity was poor. The structural analysis of LPK found that both Leu and Lys amino acids had more alkyl groups, which leaded to the peptide being far away from the active sites, resulting in weaker ACE activity of LPK (Abdelhedi et al. 2017). Molecular docking simulation is an important tool for determining the hypotensive effects of LPK and PRP (Abdelhedi et al. 2017;Mirzaei et al. 2019).

Antioxidant in Physiochemical Assays
To evaluate the antioxidant activities of LPK and PRP, DPPH (1,1-Diphenyl-2-Picrylhydrazyl), superoxide anion, and hydroxyl free radical scavenging assays were investigated. The results were shown in Fig. 6a-c. Both LPK and PRP showed obvious DPPH scavenging activity, and the scavenging activity of PRP was significantly higher than that of LPK at a concentration of 2-10 mg/ml, and their IC 50 value was 13.38 and 5.82 mg/ml (P < 0.05), respectively. The superoxide anion scavenging activity of PRP was stronger than that of LPK in the concentration range of 2-8 mg/ml. The hydroxyl radical scavenging activity of LPK and PRP was very significant. When the concentration was 4 mg/ml, the hydroxyl radical scavenging activity of PRP reached 92.96 ± 5.2%. The IC 50 values of PRP and LPK were 1.41 and 1.20 mg/ml, respectively. These results suggested that both LPK and PRP had certain antioxidant activities in vitro. Reactive oxygen species (ROS) include various unstable free radicals, such as superoxide free radicals (·O 2− ), hydroxyl free radicals (OH·), peroxy free radicals, etc. When too many free radicals are produced in the body, oxidative stress will be triggered (Brown and Griendling 2015). Oxidative stress and associated oxidative damage are mediators of cardiovascular disease, and elevated blood pressure is largely due to excess production of ROS (Sedeek et al. 2009;Touyz 2004). Therefore, antioxidant activity was an assistant factor in reducing hypertension. LPK and PRP have dual bioactivity of ACE inhibition and antioxidant, which were benefit to reduce blood pressure.

Cytotoxic Effect
The cytotoxic effects of the tripeptides LPK and PRP on RAW264.7 cells were firstly investigated and the results were shown in Fig. 7. The cell viability was not significantly decreased when the concentration of the tripeptides in the culture medium was in the range of 0.125 to 0.5 mg/ml, and it was even higher when the PRP concentration at 0.5 mg/ ml, that result indicated the two tripeptides were safe and no cytotoxic effects.  5 ACE combined with LPK a (green sticks) and PRP b (bule sticks). The tertiary structure of the protein is shown in pale-green cartoons, key residues are shown in cyan sticks, LPK and PRP are shown in green and blue respectively. Limon dash represents hydrogen bonds

Antioxidant in Cellular Assays
The cellular antioxidant assay involves the cell damage with ROS and the protection with antioxidant peptides (Wolfe and Liu 2007). RAW264.7 cell line is widely used as an antioxidant cell model (Li et al. 2019 Fig. 9. In the normal culture conditions, the cell viability was 100%, when the cell was treated with H 2 O 2 (1.4 mM) for 4 h, the cell viability was about 65%, it indicated a partial of cells was damaged to die by H 2 O 2 . While the cell was cultured with the tripeptides for 24 h and then added H 2 O 2 (1.4 mM), the cell viability increased. Especially, PRP treatment at the concentration 0.5 mg/ml allowed the cell viability significantly increase up to 85%, this result was attributed to the antioxidant activity of PRP. In contrast, the cell viability was not significantly increased when LPK was added to the culture; it indicated that LPK was weak to protect the cells from oxidative-damaging.
The antioxidant activities of the tripeptides were attributed to their capability to scavenging ROS. RAW264.7 cell was stain with the fluorescent probe, and the fluorescence intensity indicated the ROS level. As shown in Fig. 10. In this study, the increase of PRP concentration leaded to ROS levels decrease, thus PRP could remove ROS to increase the cell viability. LPK was weak to remove ROS in comparison. The results indicated that PRP was an excellent antioxidant peptide. A similar study was reported that peptides isolated from salmon protein hydrolysate were significantly downregulated ROS levels in HUVECs (Wu et al. 2021).

Mechanism Analysis of ACE and ROS Influencing Hypertension
In this present investigation, it was found that the peptides LPK and PRP derived from the enzymatic hydrolysate of S. nudus had ACE inhibition and antioxidant activity. Mechanism of ACE and ROS influencing hypertension was analyzed in Fig. 11. ACE increases blood pressure by converting inactive angiotensin-I (Ang I) into effective vasoconstriction angiotensin-II (Ang II) (Cai and Harrison 2000;Zalba et al. 2001). Therefore, inhibition of ACE activity can reduce Ang II and allow vasodilation to lower blood pressure. Both LPK and PRP have ACE inhibitory activity, which have the potential to become antihypertension drugs.
In a healthy vascular system, there is an oxidant/antioxidant balance, the mentioned oxidants are mostly ROS produced by vascular smooth muscle cells. These ROS are some unstable free radicals, such as superoxide free radicals (·O 2− ), hydroxyl free radicals (OH·), peroxy free radicals, etc. superoxide (·O 2− ) can rapidly react with nitric oxide (NO) in blood to form a strong oxidant peroxynitrite anion (ONOO − ), which is a powerful chemical anion to cause tissue damage and endothelial dysfunction, in turn lead to hypertension (Cifuentes and Pagano 2006;Harrison et al. 2006;Zalba et al. 2001). Therefore, the depression of ROS level is beneficial to lowering blood pressure. NAD(P)H oxidase, found in membrane of vascular smooth muscle cell, can be activated to produce ROS (Cifuentes and Pagano 2006). Ang II is the activator of NAD(P)H oxidase, therefore Ang II reduction could lead Fig. 8 The effect of H 2 O 2 on RAW264.7 cells viability A Cell morphology treated with 0 (a), 1 (b), 1.2 (c), 1.4 (d), 1.6 (e), 1.8 (f) mM H 2 O 2 for 4 h. B Cells viability presented as means ± SD (n = 3), **Represented p < 0.01, and *presents p < 0.05 compared with control  to ROS decreasing. Under chronic hypertension associated with oxidative stress, the oxidant/antioxidant balance is broken, two NAD(P)H oxidases, Nox1 and Nox4, belonging NOX family, were found up-regulation (van Esch et al. 2008, Bomback andToto 2009). Both LPK and PRP can scavenge ROS and meanwhile inhibit its excessive production by inhibiting the generation of Ang II. Thus the tripeptides could be applied as ACE inhibitors, ROS scavengers, and NOX inhibitors to reduce ROS bioavailability, which were synergistic interactions to antihypertention Mazumdar et al. 2021).

Conclusion
In this experiment, the conditions for hydrolysing S. nudus proteins were optimized by RSM with a BBD design. The optimal conditions were as follows: enzymolysis temperature was 52.5 °C, time 4 h, typsin dosage 0.33%. The ACE inhibition rate was 60.78 ± 6.50%, which corresponded well with the predicted value. The hydrolysate of S. nudus was purified by ultrafiltration and gel chromatography, novel tripeptides (Leu-Pro-Lys and Pro-Arg-Pro) were identified by LC-MS/MS. In vitro physicochemical experiments showed that the tripeptides had dual bioactivity of ACE inhibition and antioxidant. Molecular docking results suggested that the tripeptides mainly interacted with ACE by hydrogen bonds. LPK and PRP could inhibit the activity of ACE and block the production of Ang II. Cell culture results suggested the two tripeptides had antioxidant effects and could scavenged excess ROS. Meawhile, the tripeptides scavenged superoxide free radicals to prevent the formation of ONOO-and protect blood vessels, which were benefit to lowering blood pressure. In a conclusion, the two tripeptides had the potential to be used as antihypertension drugs and functional foods.