Isolation Of Anti-tumor Peptides Derived From Spirulina Platensis And Study On Its Anti-tumor Activity

To screen antitumor peptides from Spirulina, this paper first compares the protein extraction rate between the two methods, salt-extraction and water-extraction based on the pretreatment methods of coalition of multigelation, homogeneity and ultrasonic-extraction. Then Spirulina polypeptides are prepared by single, double and triple enzymolysis process of Spirulina proteins by using different enzymes. Further separation and purification of proteolytic peptides are carried by ultrafiltration and gel chromatography. The whole research uses MTT method to evaluate the anti-tumor activities of polypeptide components on five kinds of cancer cells in vitror, and establishes nude mice bearing human hepatoma Hep G-2 xenograft model to evaluate the anti-tumor activity in vivo. Mass spectrometry is used to analyze and identify the screening anti-tumor peptides, and finally we synthesize the corresponding peptides and verify its antitumor activity. The main results of this study are as follows:1. By using methods of coalition of multigelation, homogeneity and ultrasonic to pretreat Spirulina powder, the extraction rate of the protein in water can reach 84.80%, which is better than extraction in saline solution. So is the protein solubility.2. Spirulina polypeptides are prepared by enzymatic hydrolysis of Spirulina proteins by using trypsin, pepsin, alkaline protease and papain respectively, using alkaline protease and papain together, and using trypsin, pepsin and chymotrypsin to simulate digestive process of gastrointestinal tract in vitro. Results of the degree of hydrolysis and anti-proliferation effect suggest that there was non-linear correlation between the antitumor activity and degree of hydrolysis.3. Decolorization process would lower the antitumor activity of hydrolytic components. It can be said that exist of the chromophore or we say the integrity of chromophore structure may be one of the factors which would reduce antitumor activity of the Spirulina hydrolyzed peptides.4. After ultrafiltration, trypsin hydrolysis peptide was separated as T>10K、T5-10K、T3-5K and T0-3K. T0-3K was chosen to be purified by Sephadex G-15 as 7 components, T1 to T7. Among them, T5 can inhibit the the growth of SGC-7901 in vitro significantly, its IC50 value is 32.37μg/m L; T3 and T4 can inhibit both the growth of SGC-7901 and HT-29 in vitro significantly, their IC50 values are 46.43μg/m L, 76.11μg/m L and <31.25μg/m L, 31.25μg/m L, respectively.5. After ultrafiltration, Alcalase2.4 hydrolysis peptide was separated as A>10K、A5-10K、A3-5K and A0-3K. A0-3K was chosen to be purified by Sephadex G-15 as 5 components, A1 to A5. Among them, A1 has significant anti-prolifiration effect on Hep G-2、SGC-7901 and A549, the value of IC50 is 47.67μg/m L, 84.40μg/m L and 61.22μg/m L respectively; A2 can inhibit the growth of SGC-7901 in vitro significantly, the value of IC50 is 55.77μg/m L; A3 has significant anti-prolifiration effect on MCF-7、Hep G-2 and HT-29, the value of IC50 is 64.59μg/m L、61.65μg/m L and 43.05μg/m L respectively; Both A4 and A5 have significant anti-prolifiration effect on Hep G-2, their IC50 values are 79.44μg/m L and 69.54μg/m L, respectively.6. After ultrafiltration, papain hydrolysis peptide was separated as P>10K、P5-10K、P3-5K and P0-3K. P0-3K was chosen to be purified by Sephadex G-15 as 7 components, P1 to P7. Among them, P1 and P5 have good anti-prolifiration activities on Hep G-2, their IC50 values are 64.94μg/m L and 78.62μg/m L, respectively; P4 and P6 can significantly inhibites the growth of A549, their IC50 values are all less than the minimum dose of experiment concentration, 31.25μg/m L. Peptide from P4 with m/z = 808.418 is identified by MALDI-TOF/TOF-MS, its sequence is YGFVMPRSGLWFR(C�'N), molecular weight is 1614.8130 Da. The corresponding Synthetic peptide has good anti-prolifiration effect on A549、Hep G-2 and SGC-7901, their IC50 values are 104.05μg/m L、188.23μg/m Land 247.32μg/m L.7. After ultrafiltration, double-enzyme hydrolysis peptide was separated as D>10K、D5-10K、D3-5K and D0-3K. D0-3K was chosen to be purified by Sephadex G-15 as 6 components, D1 to D6. Among them, D1 has significant anti-prolifiration effect on MCF- 7, Hep G 2 and A549, their IC50 values are <31.25μg/m L、49.36μg/m L and 79.32μg/m L, respectively; D2 inhibites both the growth of Hep G-2 and A549 significantly, the IC50 values are 96.06μg/m L and 99.23μg/m L;D3 inhibites the growth of MCF-7 and HT-29 in vitro, their IC50 values are both less than the minimum dose concentration, 31.25μg/m L; D4 has significant anti-prolifiration effect onA549 and HT-29 in vitro, their IC50 values are 45.80μg/m L and 89.20μg/m L, respectively; D5 can inhibites the growth of SGC-7901、A549 and HT-29 in vitro, the IC50 values are 87.52μg/m L、61.14μg/m L and 34.65μg/m L, respectively. And D4 is identified as a single peptide which has 8 animo acids and the sequence is KFLVLCLRand its molecular weight is 991.561 Da. The corresponding synthetic peptide has a little antitumor activity while peptide LCLRsynthesized according to segmental sequence of D4 has good anti-prolifiration effect on Hep G-2 and HT-29, their IC50 values are 264.86μg/m L and 224.74μg/m L, respectively. Besides, peptide with molecular weight 970.599 Da and amino acid sequence AGGASLLLLRfrom D1, and peptide with molecular weight 808.481 Da and amino acid sequence LAGHVGVRfrom D5 are identified. But both the synthetic peptides have little anti-prolifiration effect on five kinds of tumor cells, which is far lower than their source components’ antitumor activity.8. After ultrafiltration, triple-enzyme hydrolysis peptide was separated as Tr>10K、Tr5-10K、Tr3-5K and Tr0-3K. Tr0-3K was chosen to be purified by Sephadex G-15 as 4 components, Tr1 to Tr4. Among them, Tr1 inhibites the growth of MCF-7 significantly, the IC50 value is 60.12μg/m L;Tr2 anti-prolifiration effect on MCF-7、Hep G-2 and SGC-7901, their IC50 values are <31.25μg/m L, 36.42μg/m L and 48.25μg/m L, respectively. Peptide from Tr1 with m/z = 935.556 is identified, its molecular weight is 935.556 Da, and the amino acid sequence is HVLSRAPR. The corresponding Synthetic peptide has significant antitumor activity on HT- 29 in vitro, the IC50 value is 99.88μg/m L.9. Results of antitumor activity experiment in vivo indicate that antitumor peptide D1 can inhibite the growth of Hep G-2 transplanted tumor in nude mice, and there’s dose-effect relationship between dose of D1 and its antitumor activity. However, the activity of D1 is lower than the positive control group in the certain scope of doses in vivo while they are quite the same in vitro. Campared to 5-FU, D1 has little effect on the weight of nude mice and that means the toxicity of D1 was much lower than positive control.