Study On Fingerprint Of Amino Acids In Konjac Mannatide

Mannatide is one of the biological response modifiers developed by Chinese scientists, which can be used for increasing immunity, anti-anemia, anti-tumor, etc.. Market mannatide is extracted from fermentation products of α-hemolytic Streptococcus strains 33, which is seperated from human throat. Hydrolysis of mannatide will produce free amino acids, of which can be detected by the amino acid fingerprint to reflect its quality. Additionally, as the by-product during the production of konjac flour, konjac flying powder’ quality is light, particles is small, occupying 38% of the konjac flour quality. Recent researches have discovered that Konjac flying powder contain abundant sugars, proteins, minerals, fiber, and other types of nutrients, affording itself very high development values, as well as a good source of obtaining mannatide.In this research, fingerprint of amino acids in konjac mannatide were evaluated by high performance liquid chromatography, which is aim to control the quality of konjac during mannatide production and providing theoretical data. The Main contents of this paper can be generalized as follows:As the raw material, konjac flying powder from different places of Shaanxi were applied for mannatide preparation. First, the extraction of polypeptide from Konjac was based on the enzymolysis method, then the crude extracts of polypeptide were treated separately utilizing Sevage method, after which mannatide was collected with further purification by DEAE-52 cellulose column and Sephadex G-100 column chromagraphy.The results indicated that casein phosphopeptide was used as a standard to plot standard curve, the linear regressione quationis is: y=0.992x﹢0.00233,R2 =0.9995,showed good linear relations. The yields among 10 batches of konjac polypeptide were in the range of 13.96%~14.71%. After separation by DEAE-52 cellulose column chromatography, number 29 to 42 cuvettes of eluent were collected, and separated by Sephadex G-100 column chromatography, number 7 to 12 and number 22 to 34 cuvettes of eluent were collected, from which isolated konjac mannanpeptide was obtained.The fingerprints of 10 batches of amino acids in konjac mannatide were also evaluated. Using the Pre-column derivatization-HPLC, Phenylisothiocyanate( PITC) was employed as derivatizing agent, mobile Phase:( A)acetonitrile-water(60:40),(B):0.1mol/L acetate-acetonitrile(93:7,adjust p H to 5.9 with acetic acid); Separation was performed on Diamonsil C18(250 mm×4.6 mm, 5μm) analytical column; The column temperature was at 30 oC; gradient elution with the flow rate of 1.0m L/min; The Fluorescence detecting wavelength was set at 254nm; Incoming sample amount was 20μL; Time analysis was 40min; gradient elution;The retention time and area of common peaks were no significant changes, the relative standard deviation(RSD) of precision and reproducibility was not more than 3%.The test solution was stable in 24 hours. Use the chroromatographic fingerprint similarity evaluation system A software to analyze data, the similarities of all the samples no less than 0.9, 10 samples were highly similar.Twelve common peaks could be found on the HPLC fingerprints, there are Serine,Glycine,Argnine,Alanine,Tyrosine,Cystine,Valine,Methionine,Phenylalanine,Isoleucine,Leucine,Lysine.The results demonstrated that its quality is better.In order to compare the quality of konjac mannan mannatide, amino acids of mannatide injection, Traditional Chinese Medicine Injection Fingerprint Technology Requirements(provisional) as the standard, for the first time, the pomegranate peel polyphenols HPLC fingerprint were studied to provide a sensitive and specific method for controlling the quality of mannatide injection.The fingerprints of amino acids in mannatide injection for ten samples which come from two factories were both established,twelve common peaks in the fingerprints of mannatide injection were determined. the similarity count of 10 patches of injection was in the range of 0.935~0.999,survey of fingerprint methodology and similarity evaluation results meet the national requirements of traditional Chinese medicine fingerprint, the quality was more stable.