Study On Purification And Anti-cancer Effects Of Peptides Derived From Porphyra Haitanesis

To obtain the anti-tumor peptides from Porphyra haitanesis, this paper compared the protein extraction rate between two methods, repetitive freeze-thawing used alone and the combination of repetitive freeze-thawing method and ultrasonication. Then the extracted proteins were hydrolyzed by papain, trypsin and pepsin. Response surface methodology was applied to optimize condition of the three kinds of enzyme’s hydrolyzation. The obtained proteolytic peptides were separated and purified by ultrafiltration and gel chromatography, and the anti-tumor activities were guided by MTT method. The obtained proteolytic peptides with good anti-tumor activities were analyzed and identified the sequence by the mass spectrometry method, then synthesized the corresponding peptides, verified its anti-tumor activities and explored the primary anti-tumor activities mechanism. The results as follows:（1） Using the methods of repetitive freeze-thawing used alone and the combination of repetitive freeze-thawing method and ultrasonication to extract protein, the extraction rate of protein is 25.58% and 40.39%, respectively, the content of protein is 40.75% and 48.66%, respectively. So the combination of repetitive freeze-thawing method and ultrasonication was better.（2） Establishing responds surface models of papain, trypsin and pepsin. The factors including p H value, temperature and [E/S] were investigated in terms of the degree of hydrolysis. Among them, the optimum conditions of papain were as follow: p H 7.02, temperature 61.4 ℃, [E/S] 3%, under this condition, degree of hydrolysis was 50.28%; the optimum conditions of trypsin were as follow: p H 8.44, temperature 44.52 ℃, [E/S] 3%, under this condition, degree of hydrolysis was 34.26%; the optimum conditions of pepsin were as follow: p H 2.06, temperature 37.4 ℃, [E/S] 2%, under this condition, degree of hydrolysis was 8.09%.（3） After ultrafiltration, papain hydrolysis peptides, trypsin hydrolysis peptides and pepsin hydrolysis peptides were separated 11 components as P_{>10 K}, P_{5-10 K}, P_{0-3 K}, T_{>10 K}, T_{5-10 K}, T_{3-5 K}, T_{0-3 K}, S_{>10 K}, S_{5-10 K}, S_{3-5 K} and S_{0-3 K}, respectively. Determining 1 mg/m L component to the growth of MCF-7, Hep G-2, SGC-7901, A549 and HT-29 in vitro. Among them, P_{>10 K} had strong anti-proliferation effects on Hep G-2 and SGC-7901 in vitro, the inhibition rate up to 100%, and the the inhibition rate of A549 was 82.33%. P_{5-10 K} could inhibit the the growth of MCF-7 and HT-29, the inhibition rate were 88.56% and 92.77%, respectively. P_{0-3 K} could inhibit the the growth of MCF-7, Hep G-2 and HT-29 in vitro significantly, the inhibition rate up to 100%, T_{>10 K} had strong anti-proliferation effects on MCF-7 and SGC-7901, the inhibition rate were 98.1% and 88.83%, respectively. T_{5-10 K} could inhibit the the growth of MCF-7 and SGC-7901, the inhibition rate were 96.81% and 91%, respectively. T_{3-5 K} had a good anti-proliferation effect on A549, the inhibition rate was 72.41%. T_{0-3 K} could inhibit the the growth of MCF-7, A549 and HT-29, the inhibition rate were 74.44%, 77.06% and 77.52%, respectively. S_{>10 K} had strong anti-proliferation effects on MCF-7, Hep G-2, SGC-7901 and HT-29, the inhibition rate were 98.65%, 99.67%, 98.28 and 87.91%, respectively. S_{5-10 K}, S_{3-5 K} and S_{0-3 K} could not inhibit the the growth of the five kinds of cells in vitro significantly. Considering the bioactivity and molecular weight, P_{0-3 K} and T_{0-3 K} were selected to isolation and purification.（4） P_{0-3 K} was chosen to be purified by Sephadex G-15 as components which named P1, P2 and P3. Among them, P2 had strong anti-proliferation effects on MCF-7 and Hep G-2, its IC50 value were 63.64 μg/m L and 59.09 μg/m L, respectively, and the inhibition rate of LO2 was 29.7% when the concentration of P2 was 500 μg/m L; P3 could inhibit the the growth of Hep G-2 and A549 in vitro significantly, its IC50 value were 209.09 μg/m L and 272.67 μg/m L, respectively, the inhibition rate of LO2 was 16.77% when the concentration of P3 was 500 μg/m L. IC50 value of 5-fu on MCF-7, Hep G-2 and A549 were 195.45 μg/m L, 122.72 μg/m L and 201.79 μg/m L, respectively. Peptide from P2 with m/z=1846.8613 was identified by MALDI-TOF/TOF-MS, its sequence was QTDDNHSNVLWAGFSR, The corresponding synthetic peptide had a good anti-prolifiration effect on Hep G-2, its IC50 value was 271.63 μg/m L（5） After ultrafiltration, T_{0-3 K} was separated as T1, T2, T3 and T4 by Sephadex G-15. Among them, T1 had good anti-proliferation effects on MCF-7, Hep G-2, SGC-7901 and A549, its IC50 value were 280.28 μg/m L, 316.95 μg/m L, 285.18 μg/m L, 191.61 μg/m L, respectively, the inhibition rate of LO2 was 12.2% when the concentration of T1 was 500 μg/m L; T2 could inhibit the the growth of MCF-7 and HT-29 in vitro significantly, its IC50 value were 281.71 μg/m L and 206.44 μg/m L, respectively, the inhibition rate of LO2 was 25.7% when the concentration of T2 was 500 μg/m L. T3 had a good anti-proliferation effect on HT-29, its IC50 value was 231.92 μg/m L, the inhibition rate of LO2 was 20.24% when the concentration of T3 was 500 μg/m L. IC50 value of 5-fu on MCF-7, Hep G-2, SGC-7901, A549 and HT-29 were 195.45 μg/m L, 122.72 μg/m L, 201.79 μg/m L, 226.25 μg/m L and 413 μg/m L, respectively. Peptides from T1 with m/z=1280.676 and m/z=1677.879 were identified by MALDI-TOF/TOF-MS, its sequence were VPGTPKNLDSPR and MPAPSCALPRSVVPPR, respectively. The corresponding synthetic peptide VPGTPKNLDSPR had good anti-prolifiration effects on MCF-7 and Hep G-2, its IC50 value were 200.97 μg/m L and 276.85 μg/m L, respectively. The corresponding synthetic peptide MPAPSCALPRSVVPPR had anti-prolifiration effects on the five kinds of cancer cells in vitro, but the activities were not as good as corresponding synthetic peptide VPGTPKNLDSPR.（6） Using Flow Cytometry to explore primary anti-tumor activities mechanism of the corresponding synthetic peptide VPGTPKNLDSPR which inhibited growth of MCF-7. The result of PI showed that cells proportion in G0/G1 increase gradually under the concentration gradient of 125 μg/m L, 250 μg/m L, 500 μg/m L, the cell retardation in G0/G1. The result of Annexin V-FITC/PI showed that quantity of early apoptotic cells increased gradually. The total quantity of apoptotic cells increased from 1.2%（control） to 7.2%, 16.1% and 19.2% under the concentration gradient of 125 μg/m L, 250 μg/m L, 500 μg/m L. The corresponding synthetic peptide VPGTPKNLDSPR inhibited growth of cancer cells by inducing cell apoptosis.