Study On Purification And Anti-cancer Effects Of Peptides Derived From Perilla Frutescens

Perilla frutescens,one of medicinal and edible plants promulgated by the Chinese Ministry,is notable as an important source of edible oil obtained from its seeds.At present,Perilla frutescens is mainly used in oil squeezing while its abundant protein hasn't been fully utilized with the perilla cake being used as animal feeds.In order to improve the utilization level of perilla seeds,promote the comprehensive development of perilla protein,the study used perilla cake as the raw material to prepare perilla protein and perilla polypeptide,then conducted researches on the functional properties of perilla protein and the antioxidant and anticancer activity of perilla polypeptide.Based on that,perilla yogurt has been made from fresh milk and perilla polypeptide.The main results of study were as follows:(1)Response surface methodology(RSM)was used to optimize the ultrasound-assisted extraction parameters of perilla protein.The optimum conditions were an ultrasonic power of200 W,extraction time of 32 min,and a liquid-to-solid ratio of 10:1.Under these conditions,protein yield was 26.7%.With a comparison of the functional properties of perilla protein and soy protein,the results showed that the functional properties of perilla protein(water solubility index 56.1%,oil holding capacity 1.47 g/g,emulsifying activity 61.13 m~2/g,water absorption capacity 33.3%,foaming ability 62.7%)were suitable to be used in the food industry.(2)Perilla protein was hydrolysed by using Alcalase,the ultrafiltration isolates of hydrolysed mixture were PSP1,PSP2 and PSP3.It was found that the antioxidant capacity of PSP3 was the strongest in DPPH radical scavenging ability.Meanwhile,Sephadex G-15 gel chromatography was used to isolate and purify PSP3 and obtained PSP3a,PSP3b and PSP3c.DPPH radical scavenging ability showed that PSP3c had the strongest antioxidant capacity.The RP-HPLC analysis of PSP3c displayed a single peak,which indicated its higher purity.PSP3c was also detected with mass spectrograph and sequence analysis.Its molecular weight and sequence was 716.77 Da and Ala-Ser-Pro-Gly-Leu-Trp-Ser respectively.(3)HepG2 cells were selected as test subject to investigate the apoptosis effect of COGP2a on tumor cells.The MTT examination results showed:PSP3c has a certain inhibitory effect on HepG2 cells.The inhibitory effect reached 90%when the density of PSP3c was 100?g/mL.After being treated with PSP3c,the HepG2 cells were stained by Hoechst 33258.Obvious nuclear chromosome condensation,nuclear fragmentation and strong blue fluorescence were observed by fluorescence microscope.In addition,the various stages of HepG2 cells stained by Annexin V-FITC and PI were analyzed by flow cytometry.With the increase of the PSP3c density,the normal cells were on decrease while the amount of early apoptotic cells,late apoptotic cells and dead cells increased.When the density amounted to 100?g/mL,the apoptotic rate was 52.5%,which further proved PSP3c had a certain inhibitory effect on HepG2 cells.HepG2 cells treated with PSP3c demonstrated an increasing expression of Caspase-3 protein which suggested that PSP3c could activate the apoptotic pathway.(4)The antitumor and antioxidant activity of PSP3c in vivo was appraised through establishing the liver cancer model by transplanting H22.PSP3c's tumor inhibition rate was 69.5%when its concentration was 20 mg/mL,which showed PSP3c's strong anticancer activity in the mouse subjects.Then,the H22 mouse subjects were tested in the following aspects:thymus index,pancreas index,the number of white blood cells and the number of lymphocytes.The results showed that all those indexes had effectively increased.PSP3c did no harm to the immune system of mouse subjects.Instead,it was helpful in the recovery of immune organs damage caused by the cancer cells and inhibited the duplication of cnacer cells.PSP3c's antioxidant activity were evaluated through testing the changes of GSH-Px and SOD in H22 mouse subjects'blood and liver.At the different concentration of PSP3c,the amount of MDA in H22 mouse subjects'blood and liver had decreased variably,but the activities of GSH-Px and SOD had rebounded correspondingly.It turned out that PSP3c is able to increase the antioxidant activity and helpful to inhibit the cancer cells'duplication.(5)Fresh milk and Perilla polypeptide(PSP3c)were used to make a special yogurt.The orthogonal experiments results showed that the optimized fermentation conditions were:sugar6%,inoculums 0.3%,PSP3c 8%.The antioxidant activity tests revealed that the special yogurt exhibited a higher expression than traditional yogurts.