“Food Science”: Preparation and antihypertensive activity of angiotensin-converting enzyme inhibitory peptide derived from pumpkin seed protein

Hypertension is the most common chronic cardiovascular disease, which is closely related to the complications of various human organs and seriously endangers human health.

At present, clinical antihypertensive drugs angiotensin-converting enzyme (ACE) inhibitors such as captopril, alapril, enalapril and lisinopril are effective, but can cause side effects such as vomiting and palpitations. In recent years, food-derived bioactive peptides have attracted great attention due to their nutrition and safety.

Pumpkin Seed Protein: Pumpkin seeds contain 30% to 40% protein, rich in amino acid composition, and are a good vegetable protein resource. At present, there are relatively few studies on ACE inhibitory peptides derived from pumpkin seed protein, and the possible ACE inhibitory molecular mechanism of pumpkin seed peptides is not clear enough.

Therefore, alkaline protease was used to hydrolyze pumpkin seed protein to release ACE inhibitory peptide, and ACE inhibitory peptide was obtained by membrane separation technology and its structure was identified by mass spectrometry. Kinetic and molecular docking methods were used to study the blood pressure-lowering activity and enzyme inhibition mechanism of ACE inhibitory peptides, in order to provide theoretical basis for the development of pumpkin seed blood pressure-lowering peptides.

1. Hydrolysis of pumpkin seed protein and activity analysis of hydrolyzate membrane separation components

The degree of hydrolysis of pumpkin seed protein was positively correlated with the hydrolysis time. The degree of hydrolysis increased rapidly within 1 h after the hydrolysis started, and the degree of hydrolysis reached 7.99%; the degree of hydrolysis increased by 2.99% within 60 to 200 min, and the hydrolysis reaction rate was significantly slower, and the rate of hydrolysis was significantly slower at 4 h. The post-hydrolysis degree reached 11.23%, and the active peptides in the pumpkin seed protein were fully released under the action of alkaline protease.

It can be seen that at the mass concentration of 1 mg/mL, the PPH ACE inhibitory activity also varies with the molecular weight of the hydrolyzate. The ACE inhibitory activities of PPH and peptide components below 1 kDa were higher, and there was no significant difference in ACE inhibitory activity between the two (P>0.05), which were 46.81% and 46.22%, respectively; except for the 3-5 kDa component, ACE inhibited The activity gradually decreased with the increase of the molecular weight of PPH, and the 5-10 kDa component had the lowest ACE inhibitory activity, with an inhibition rate of 33.74%.

The hypotensive activity of PPH is shown in Fig. 1C. After SHRs were administered with PPH (100 mg/kg mb), PPH and PPH components below 1 kDa exhibited similar hypotensive effects at 4 h after intragastric administration, and both decreased The systolic blood pressure of SHRs was about 14.80 mm Hg, while the PPH component below 1 kDa showed the best blood pressure-lowering effect after gavage for 6 h, reducing the systolic blood pressure of SHRs by about 21.42 mm Hg, which was lower than that of the 1 kDa PPH component. The antihypertensive activity is better than that of PPH; although the PPH component below 1 kDa shows a sustained antihypertensive effect, it is still inferior to the antihypertensive drug captopril.

2. Structural identification of pumpkin seed protein peptides

In order to determine the structure of the peptides that may cause the activity, HPLC/Q-TOF-MS/MS was used to identify the peptide composition of pumpkin seed protein in the PPH fraction below 1 kDa. On the basis of obtaining total ion chromatograms and first-order mass spectra of PPH components below 1 kDa, PEAKS Studio software was used to analyze and compare the UniProt database, and 9 peptides were identified from PPH components below 1 kDa , respectively Leu-Leu-Val (LLV), Leu-Val-Phe (LVF), Leu-Thr-Pro-Leu (LTPL), Ser-Val-Leu-Phe (SVLF), Leu-Leu-Pro-Gln (LLPQ), Met-Leu-Pro-Leu (MLPL), Leu-Leu-Pro-Gly-Phe (LLPGF), Val-Leu-Leu-Pro-Glu (VLLPE), Arg-Phe-Pro-Leu-Leu (RFPLL).

In addition, by trying to search the bioactive peptide database, it was found that none of the identified 9 pumpkin seed peptides appeared in these ACE inhibitory peptide or blood pressure lowering peptide data, only similar blood pressure lowering peptide sequences were found, such as LLY, LVY, LVE, LLP, LLF, etc. (Search databases: http://bis.zju.edu.cn/biopepdbr/index.php, http://pepbank.mgh.harvard.edu, http://www.nipgr.ac.in/ PlantPepDB/), indicating that new ACE inhibitory peptides were discovered from PPH in this study.

3. ACE inhibitory activity and blood pressure lowering activity of pumpkin seed peptide

As shown in Figure 3A, the ACE half-inhibitory concentrations (IC50) of the nine polypeptides RFPLL, VLLPE, LLPGF, MLPL, LLPQ, SVLF, LTPL, LVF, and LLV were 0.94, 16.57, 0.48, 0.36, 5.32, 7.11, 5.07, 0.17, 3.16 mmol/L, among which the IC50 of LLPGF, MLPL, LVF and RFPLL were all lower than 1 mmol/L, showing high ACE inhibitory activity.

The antihypertensive activities of pumpkin seed peptides LLPGF, MLPL, LVF and RFPLL with better ACE inhibitory activity were further evaluated.

Overall, RFPLL has a good blood pressure lowering activity, systolic blood pressure decreased by 37.0 mm Hg and diastolic blood pressure by 17.0 mm Hg after gavage of SHRs for 6 h; followed by LVF, systolic blood pressure decreased after gavage of SHRs for 6 h 22.2 mm Hg, diastolic blood pressure decreased by 11.2 mm Hg; while LLPGF and MLPL had relatively poor blood pressure lowering activity; RFPLL and LVF still had certain blood pressure lowering activity after 24 h of intragastric administration of SHRs, and RFPLL and Cato The blood pressure lowering effect of Puli was similar, indicating that a better blood pressure lowering peptide was obtained from pumpkin seed protein.

4. Kinetic analysis of pumpkin seed peptides inhibiting ACE

It can be seen from Figure 4 that RFPLL is a mixed-type inhibitor of ACE. At low concentration (0.5 mmol/L) RFPLL is a competitive inhibitor to ACE, and a higher concentration (1 mmol/L) shows a non-competitive inhibitory effect; The reciprocal straight line approximately intersects the y-axis, indicating that its inhibition mode to ACE may be competitive inhibition, while LLPGF and LVF may be non-competitive inhibition types.

5. Molecular docking between pumpkin seed peptide and ACE

The results show that RFPLL, LLPGF, MLPL and LVF can be well docked to the active center residues of ACE. Among them, RFPLL and ACE residues Tyr523, Glu162, Asp377 form three hydrogen bonds, and through O31, O28 Chelated Zn 2+ and obtained a lower electrostatic potential energy of -231.64 kJ/mol, indicating that RFPLL can exert inhibitory activity by binding to the ACE active site S1, S1′ and the triplet catalytic site; Residues His353, Ala354, Tyr520, Lys511, Gln281 form 9 hydrogen bonds, and the distance between O2 and Zn 2+ is only 2.78 Å.

in conclusion

The results showed that: 1) PPH components below 1 kDa had higher blood pressure lowering activity, and could reduce the systolic blood pressure of SHRs by about 21.42 mm Hg after gavage for 6 h; 2) Pumpkin seed peptides RFPLL, LLPGF, MLPL and LVF had better blood pressure lowering activity. The ACE inhibitory activity of the SHRs was lower than 1 mmol/L, and the RFPLL and LVF had better blood pressure lowering activities. After 6 h of intragastric administration, the systolic blood pressure of SHRs was reduced by 37.0 mm Hg and 22.2 mm Hg, respectively, and the SHRs were reduced. Diastolic blood pressure decreased by about 17.0 mm Hg and 11.2 mm Hg; 3) RFPLL, LLPGF, MLPL and LVF can well dock the active center of ACE, RFPLL is a mixed inhibitor of ACE, and MLPL is an inhibitory mode of ACE, which may be competitive inhibition , LLPGF, LVF may be non-competitive inhibitors of ACE.

In conclusion, PPH below 1 kDa is a good raw material for blood pressure lowering functional food, while RFPLL and LVF can be used as raw material for the development of blood pressure lowering functional food or medicine.