Team:SZPT-CHINA/Demonstrate
IN VITRO ACTIVITY OF AHP
DEMONSTRATE
The SZPT-CHINA team tried to lower blood pressure with food-derived antihypertensive peptides this year. Through one year’s hard working we have developed products, anti-hypertensive peptide nanocapsules and antihypertensive probiotics. We validate the viability of our products in actual work through different methods.
In the E. coli expression system, we removed the fusion tag by trypsin and obtained the antihypertensive peptide multimer by affinity chromatography. The polymer was hydrolyzed by trypsin and α-chymotrypsin to obtain active AHPs monomer. The result of in vitro activity is shown in Fig.1. The IC50 of inhibiting ACE activity is 0.1210mg/ml.
The AHPs after enzymatic hydrolysis were used as core materials, and PLGA was used as a wall material to prepare antihypertensive peptide nanocapsules. The result is shown in Fig.2. The nanocapsules have a particle size distribution between 300 and 1000 nm. We have successfully prepared hypotensive peptide nanocapsules.
It is reported that nanocapsules help to increase the sustained release and bioavailability of AHPs, and it can significantly improve the blood pressure lowering effect of AHPs. Unfortunately, due to the limited time and application of the animal use form, the hypotensive effect of AHPs in animals was not evaluated. If possible, we will further evaluate their biological effects.
The collected fermentation supernatant of lactic acid bacteria was hydrolyzed with trypsin and α-chymotrypsin to measure its in vitro activity. The results were shown in Fig.3. The IC50 of supernatant of Lactococcus lactis MG1363（LAB MG1363） transferred with pMG36e-A11Sg-AHPM was 4.8 mg/ml. Compared with the control supernatant, the IC50 result indicated that the fermentation supernatant of LAB MG1363 transferred with pMG36e -A11Sg-AHPM has much higher inhibitor activity than control group. It shows that the fusion protein with antihypertensive peptide can be secreted outside the cell, and it has a high blood pressure lowering activity after enzymatic hydrolysis.
The gastrointestinal simulation experiment was carried out on the recombinant lactic acid bacteria, and the result is shown in Fig.4. After gastrointestinal treatment 3 hours, the IC50 was 5.2mg/ml. The inhibitor activity is much higher than without treatment group(14mg/ml). The LAB MG1363 transferred with pMG36e was the control group(18mg/ml). It shows that the recombinant lactic acid bacteria can secrete antihypertensive peptides which have the effect of lowering blood pressure after gastrointestinal digestion.
As shown above, our antihypertensive probiotics have a good application prospect for lowering blood pressure. We can use it to make convenient probiotic tablets, yogurt or other forms. According to the feedback from the questionnaire, 86.8% of the respondents prefer to choose yogurt to lower blood pressure. Fig.5 shows the products prepared using our antihypertensive probiotics.
Therefore, we evaluated the viable count of yogurt and the antihypertensive effect in vitro. The results showed that the viable count was 6.8×106cfu/ml and the IC50 was 3.7mg/ml. The LAB MG1363 transferred with pMG36e was as the control group（8.9mg/ml） (Fig.6). The result shows that yogurt has the lower blood pressure function and yogurt fermented using our antihypertensive probiotics has significant blood pressure lowering function.
Although we evaluated the viable count and the in vitro activity of hypotensive probiotics, it is not enough for it. In the future, we also need to evaluate the number of viable bacteria in the shelf life and the best storage conditions, as well as bioavailability. If possible, we will use animal models to evaluate their efficacy in the body and whether they will cause other potential hazards, such as bacterial transfer.