Study On Quality Standard Of Anti-liver Fibrosis Active Extract From Periplaneta Americana

ObjectivePeriplaneta Americana extract has the function of anti-liver fibrosis effect at the previous study of the research group.Based on the previous research,this topic aims to study the anti-liver fibrosis active extract from the character,identification,examination and determination of Periplaneta Americana.The quality standard of anti-liver fibrosis active extract(ML-HB,freeze-dried powder)of Periplaneta Americana was established by research,which laid a foundation for the research and development of new anti-liver fibrosis drug of Periplaneta Americana.Methods1.The thin-layer chromatography and chemical reaction were used for qualitative identification of proteins,polypeptides,amino acids,saccharides and nucleosides in ML-HB.2.Referring to the relevant laws and regulations in the 2015 edition of Chinese Pharmacopoeia and Rules on New Drug Examination,moisture,residue on ignition,p H value,heavy metals,ethanol residues and resin residues in ML-HB were routinely examined,and it’s solubility in different solvents were studied.The molecular weight distribution of ML-HB was investigated by Tricine-SDS-PAGE electrophoresis.3.Contents of total amino acids and free amino acids in ML-HB were determined by ultraviolet-visible spectrophotometry.Compared the effects of Lowry(Folin-Ciocalteu method),BCA(2,2’-biquinoline-4,4-dicarboxylic acid disodium salt)and Bradford(Coomassie Brilliant Blue method)on the determination of polypeptide content,and to optimize the best method for the determination of polypeptide content in samples.The total sugar content was determined by sulfuric acid-phenol method,and the reducing sugar content was determined by DNS(3,5-dinitrosalicylic acid)method,and the content of polysaccharide was determined.4.Using RPLC-DAD(reversed phase liquid chromatography-diode array detector)technology to separate and analysis ML-HB,search for the optimal chromatographic conditions,and establish the RPLC-DAD fingerprint of ML-HB.The similarity analysis of 10 batches of samples were carried out by using the software of Rraditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition,and the cluster analysis was carried out by using SPSS19.0 software.Results1.In the thin-layer chromatography,the Periplaneta Americana control drug and ML-HB showed the same color spots at the corresponding positions,and the spots were clearand separating degree is satisfaction.In the Xanthoprotein reaction,the sample solution changed from light yellow to orange,and the color change was obvious,which proved that the sample contained a protein with a benzene ring.In PAS(Periodic acid-Schiff reaction),the color of the sample was purple-red and stable,with obvious color change and high sensitivity,indicating that the sample contained glycopeptides(mucopolysaccharides).The sample quickly turned yellow,good repeatability in Indoquinone reaction,which proved that the sample contained peptide components.2.The moisture content in the sample measured by Weightlessness method is between2.3‰ and 2.5‰.The amount of the residue measured by the residue ignition method is between 2.4‰ and 2.6‰.The p H value of the sample ranges from 6.57 to 6.64.The residues of heavy metals and ethanol in the samples met the requirements of Chinese Pharmacopoeia2015 edition,and the residues of resins was not detected.The solubility of sample in 0.3% tris aqueous solution was the best.Tricine-SDS-PAGE electrophoresis with urea showed that the sample was well separated and the Marker band was clear,the molecular weight of a few substances in the sample ranged from 1.2 to 45 KDa,and that of most substances was below1.2 KDa.3.The average content of free amino acids was 23.11mg/g,the average content of total amino acids was 63.54mg/g,with a good linear relationship(r=0.9995),and the methodological study met the requirements.By compared the effects of Lowry method,BCA method and Bradford method on the determination of polypeptide content,it was proved that BCA method was more suitable for the determination of polypeptide content in samples,the average content of polypeptide was 381.0mg/g,with a good linear relationship(r=0.999 3),and meeting requirements of the methodological investigation.The total sugar content was measured by sulfuric acid-phenol method was 50.50mg/g,with a good linear relationship(r=0.999 0),and meeting the requirements of the methodological investigation.The reducing sugar content was measured by DNS method was 13.49mg/g,the linear relationship was good(r=0.999 1),the methodological study met the requirements,and the average content of the calculated polysaccharides was 37.01mg/g.4.RPLC-DAD fingerprint analysis method of ML-HB was established,which was used to determine 10 common peaks,and the relatively large and stable no.5 peak was identified as the reference peak(S),the results of the methodological investigation met the requirements.The RSD values of the 10 batches of samples were within the range of 0.73%～1.49%,indicating that the fingerprint establishment method was reliable and reproducible.The peak area of RSD of 10 batches of samples ranged from 4.99% to 40.59%,indicating that therewere differences in the component contents of samples of different batches.Compared with the common mode R,the similarity was greater than 0.823.The clustering analysis results showed that S1,S2 and S3 were clustered into one category,and the rest batches were clustered into one category.Conclusion1.TLC showed clear spots and good separation,and size,location,color of the main spots of ML-HB were consistent with those of the control drug.Xanthoprotein reaction,PAS,and Indoquinone reaction have obvious color change,good repeatability,simple and rapid operation,strong specificity,and they could be used for qualitative identification of ML-HB.2.According to the inspection results,the moisture content and residue in ML-HB should not be higher than 3.0‰ and the p H value should be between 6.2 and 7.0.The residue of heavy metal and ethanol should meet the requirements of the 2015 edition of Chinese pharmacopoeia.Residual resin shall not be detected in ML-HB;The sample should be easily soluble in 0.3% Tris aqueous solution.Tricine-SDS-PAGE electrophoresis method established in this study that could detect the molecular weight distribution range of ML-HB.3.Established determination methods of amino acid,peptide and polysaccharide in ML-HB,and stipulated that the free amino acid content should not be less than 20.80mg/g,the polypeptide content should not be lower than 343.0mg/g,and the polysaccharide content should not be less than 33.0 mg/g.4.Established the RPLC-DAD fingerprint of ML-HB.The quality standard of anti-hepatic fibrosis active extract(ML-HB,freeze-dried powder)was established by studying,which laid a foundation for the research and development of new anti-live fibrosis drugs for periplaneta Americana.