The Separation And Purification Of Anti-tumor Extract And Anti-ulcerative Colitis Extract Of Periplaneta Americana

ObjectiveTumor disease is a serious hazard threatening human health, bioactive extract C-?3 from Periplaneta americana exhibited potent in vitro cytotoxicity and in vivo antitumor effects in MTT assays and animal experiments. Ulcerative colitis(UC), with the characteristics of migration in disease course, high recurrence rate,and cancerous tendency, the World Health Organization has listed it as one of the modern refractory diseases. The bioactive extract TF also from P. americana showed a promising therapeutic effect against UC in animal model experiments.To provide references and material base for the optimization of compound purification protocol and further pharmacological evaluation, P. americana crude extract was adopted as raw material to conduct the chemical investigation on peptides form the bioactive extracts CII-3 and TF.MethodsSystematic separation and purification method was adopted to determine the protocol of separating and purifying antitumor extract C?-3 and optimize it.SDS-PAGE gel electrophoresis was applied to determine the molecular weight ranges of sample C?-3. while suitable chromatographic packing and separation conditions using in gel chromatography was also explored. According to the results of SDS-PAGE gel electrophoresis experiment, the purification conditions of glucan gel separation were then investigated and optimized, followed by ion exchange column chromatography. Specifically, the ion exchange column was employed on desalted freeze-dried sample CII-3. Meanwhile, the best purification conditions for sample separation using the ion exchange column were studied.Another separation technology reversed-phase high pressure liquid chromatography (HPLC) was adopted to further purify compounds collected from the impure peaks of ion exchange column chromatography. Same as other separation and purification technologies, separation conditions of reversed-phase HPLC were also established.The content of free amino acids and proteins of TF, the anti-UC extract TF from Periplaneta americana. were determined by ninhydrin colorimetric method and Folin phenol method, respectively. Then, SDS-PAGE and Tricine-SDS-PAGE electrophoresis as well as column chromatography with glucan gels SephadexG-25 and SephadexG-50 were implemented to measure the molecular weight ranges of TF. Ultrafiltration centrifuge was used in separating the sample TF into three parts,i.e., molecular weights greater than 10KDa (composition B), molecular weights greater than 3KDa but smaller than 10KDa (composition C), and molecular weights smaller than 3KDa (composition D). Both compositions B and D were freeze-dried, stored at -80?while composition C was subjected to further separation by preparative medium pressure liquid chromatography (MPLC).Fractions collected from MPLC were freeze-dried, and the fraction collected from the first peak of MPLC was named as composition E, which was then subjected to reversed-phase HPLC analysis to explore the separation conditions.The optimized separation conditions of reversed-phase HPLC were then expanded by linear method, and preparative reversed-phase HPLC was employed for massive collecting of pure compounds, while analytical HPLC was applied to confirm the purity of isolated compounds.ResultsOutcomes of study on extract C-?3: 1. The molecular weights ranged from 3.3 KDa to 5.8 KDa, according to Tricine-SDS-PAGE electrophoresis; 2.Compositions M, N, S were obtained by SephadexG-25; 3. No satisfactory results could be found in the experiments of further separating composition M by both ion exchange column chromatography and hydrophobic protein separation column chromatography; 4. 11 fractions which were collected from corresponding single peaks of composition M subjected to reverse high pressure liquid chromatography were obtained. The mass measures of each peak were: the first peak 14.34 mg. the second peak 15.45 mg, the third peak 16.22 mg, the fourth peak 14.32 mg, the fifth peak 15.43 mg, the sixth peak 16.56 mg, the seventh peak 16.54 mg, the eighth peak 16.45 mg. the ninth peak 17.56 mg. the tenth peak 17.73 mg, the eleventh peak 17.98 mg. though none of their purity reached chromatographic pure.Outcome derived from investigation on extract TF: 1. The contents of free amino acids and proteins in TF were 15.06% and 52.8%. 2. The molecular weights ranged from 1.7 KDa to 42.0 KDa. according to Tricine-SDS-PAGE electrophoresis; 3. Compositions B, C, D were obtained by ultrafiltration centrifugin; 4. There were 8 peaks detected when further separating composition C by MPLC; 5. 3 pure compounds were obtained, named TF-2, TF-4, and TF-6. with the quantities of 100 mg, 80 mg, and 150 mg, respectively.ConclusionThe range of molecular weights of antitumor extract C?-3 was mainly 3.3 -5.8 KDa, and another small part about 25 KDa. Because of the complexity of the compositions of CII-3, MPLC was suggested to be introduced into its separation and purification in addition to glucon gel separation method and reversed-phase phase HPLC technology.The mainly constitutes of TF were proteins and peptides. With the application of systematic separation and purification method which combined with electrophoresis, ultrafiltration centrifuging, glucon gel chromatography and reversed-phase HPLC, three pure compounds TF-2, TF-4 and TF-6 were obtained from the anti-UC extract TF.