The Isolation, Purification, Activity Assay And Structure Determination Of The ACE Inhibitory Peptides From Lycium Barbarum L.

Objective: Angiotensin converting enzyme(ACE) inhibitory peptides play an important role in the regulation of blood pressure through the renin-angiotensin system and kallikrein-activated peptides in the system. ACE inhibitory peptides from food protein source for prevention and control of hypertension has become a hotspots. Our aim is to explore the isolation, purification, activity assay and structure determination of the ACE inhibitory peptides from Lycium barbarum L., and provide the theoretical basis for the preparation of the natrual ACE inhibitory peptides from Lycium barbarum L.Methods: weighed 50 g powder of Lycium barbarum L.. Alkali extraction and acid deposition method was used to isolate the crude protein from Lycium barbarum L., then used One Way ANOVA to determine the p H value of the alkali extraction process and the p H of the acid deposition process; L9(33) orthogonal experiment method was used to ensure the optimum amount of enzyme added, substrate concentration and isolation time; prepared the crude protein enzymolysis liquid of Lycium barbarum L., and tested the content and activity assay of the peptide; used the reversed phase high performance liquid chromatography(RP-HPLC) to isolate ACE inhibitory peptides from Lycium barbarum L.; furan acryloyl tripeptide(FAPGG) was used as a simulation substrate, determined the inhibition rate of ACE inhibitory peptides from Lycium barbarum L. through detecting the N-(3[2-furan] acryloyl)-phenylalanine(FAP) and double ammonia peptide(GG) generated from FAPGG hydrolyzed by ACE; the RP-HPLC method was used to purify, analyze and identificate the protein hydrolysate of Lycium barbarum L. after purified, used liquid mass(LC MALDI TOF/TOF/MS) and the secondary mass spectrometry analysis to determine the components of the protein hydrolysate of Lycium barbarum L. and then determined the structure of peptide chain consist in the purified protein hydrolysate from Lycium barbarum L.Results:(1)The p H value of the alkali extraction process is 8.5; the p H of the acid deposition process is 4.0; optimized through the orthogonal experiment, we know the conditions to isolate the ACE inhibitory peptides from Lycium barbarum L. with the highest inhibition rate: isolating 3 hours, 20% substrate concentration, and enzyme amount added to 1%. After enzymolysis the crude protein of Lycium barbarum L., we measured its peptide content is 8.5 mg/ml; the average rate of ACE inhibition steady at 90.01%, the highest can reach 99.03%.(2) Filter the isolated protein hydrolysate from Lycium barbarum L. by Sephadex G-25 and SP Sephadex C-25 of cation exchange column for isolation and purification, under the condition of 200 nm wavelength, collect six peaks of elution components, measured these six components separately, the ACE inhibitory rates are 88.62%, 87.71%, 88.62%, 89.56%, 89.87% and 88.02%, then select the component which has the highest ACE inhibition rate, used the high performance liquid chromatography for the further purification, we gain the highest activity of 13 components in the protein hydrolysate of Lycium barbarum L., measured these 13 components separately, the ACE inhibitory rates are 61.99%, 81.77%, 78.73%, 85.23%, 78.73%, 67.28%, 63.66%, 94.34%, 82.28%, 73.85%, 82.35%, 67.06% and 82.35%; and then access to the RP-HPLC once again to separate and purify the final three components, and measure the activity of the highest component to determine the ACE inhibition rate, 95.33%, 88.26% and 84.62%, respectively.(3)By using the LC MALDI TOF/TOF/MS and secondary structure of mass spectrometry, we can draw out two main amino acid peptides which have the high activity composition, mainly composed by FNPR(phe-asn-pro-arg) and WRAYGSG(trp-arg-gla-tyr-gly-ger-gly).Conclusion:(1)The average inhibition rate under optimized conditions for the base of alkali extraction and acid deposition method of ACE inhibitory peptides from Lycium barbarum L. can reach 90.01%.(2)Isolated a new strong activity of ACE inhibitory peptides from the zymolyte using ion exchange chromatography, gel filtration chromatography and reversed phase high performance liquid chromatography and other separation methods, the inhibition rate can reach 95.33%.(3)Using LC MALDI TOF/TOF MS and secondary structure of mass spectrum analysis and structure identification, its amino acid sequence were FNPR(phe-asn-pro-arg) and WRAYGSG(trp-arg-gla-tyr-gly-ger-gly).