Study On The Chemical Composition Of Cervus And Cucumis Polypeptide Injection

Cervus and Cucumis Polypeptide Injection is the exclusive original patent product of Harbin Gloria Pharmaceuticals.It is extracted from the bones of Cervus nippon Temmick and the dried and ripe seeds of Cucumis melo L.To date,its effective substances remains unclear,and the poor quality control has been the main factor that restricts its production,sales,and clinical application.In this paper,the extract of deer bone and melo semen were analyzed by NanoLC-LTQ-Orbitrap and purified by a variety of column chromatography.The obtained compounds were identified by LCMS-IT-TOF,NMR,and MALDI-TOF-MS.Meanwhile,HPLC-ELSD,gradient precipitation,and phenol sulfuric acid were used for qualitative and quantitative analysis of monosaccharides and polysaccharides in deer bone and melon seed extract,and amino acids were analyzed by amino acid analyzer.The following results are achieved:1.The progress of clinical application and pharmacology of Cervus and Cucumis Polypeptide Injection as well as the separation,purification and identification technology of polypeptides were reviewed by systematically searching domestic and foreign documents.Literature review showed that Cervus and Cucumis Polypeptide Injection is mainly used for the treatment of osteoporosis,rheumatoid arthritis,nonunion of fracture,and ankylosing spondylitis.It has good therapeutic effect and low incidence of adverse reactions.According to modern pharmacology,Cervus and Cucumis Polypeptide Injection possesses the effects of anti-inflammatory,promoting bone cell growth and inhibiting osteoporosis.As far as purification and structural identification of polypeptides are concerned,chromatography,electrophoresis,and membrane were uaually used to isolate and purify the polypeptides,and terminal degradation technique,spectrometry based on MALDI and ESI mass were used to analyze the amino acid sequence of polypeptides.2.NanoLC-LTQ-Orbitrap was used to analyze the constituents of deer bone extract,and a large number of mass spectrum signals of polypeptides were found.Forty-three polypeptide sequences were matched by retrieving the accurate molecular weight and the MS/MS spectrum information in the database.Then,a variety of column chromatography and semi-preparative HPLC were used to isolate and purify the polypeptides of deer bone extract,and the amino acid sequence of polypeptides were determined by MALDI-TOF-MS.Thirteen polypeptides and their amino acid sequences were identified from the deer bone extract.These polypeptides include:GPVGPTGPVGAAGPSGPD(Meil8 peptide,1),AGPAGPLGPLGPLGPLGPPDSYD(Mei23 peptide,2);AGPAGPKGELGSLGE(Mei 15 peptide 1,3),ninth-bit methylation modified AGPAGPKGELGSLGE(Mei 15 peptide2,4),ninth,fifteenth-bit methylation modified AGPAGPKGELGSLGE(Mei15 peptide3,5),seventh-bit vinyl-modified and ninth-bit methylation modified AGPAGPKGELGSLGE(Mei 15 peptide4,6),seventh-bit vinyl-modified and ninth,fifteenth-bit methylation modified AGPAGPKGELGSLGE(Mei 15 peptide5,7);AGPAGPKGELGSLGED(Mei 16 peptidel,8),ninth-bit methylation modified AGPAGPKGELGSLGED(Mei16 peptide2,9),ninth,fifteenth-bit methylation modified AGPAGPKGELGSLGE(Mei 16 peptide3,10),PGLGALAPYPD(Meill peptide,11),LGPLGPAGFAGPLGAD(Mei16 peptide4,12),LGPAGKEGPVGLLGL(Mei15 peptide6,13).3.NanoLC-LTQ-Orbitrap was used to analyze melon seed extract,and no polypeptide was found.A variety of column chromatography and semi-preparative HPLC were used to isolate and purify the compounds in melon seeds,and eighht compounds were identified by LCMS-IT-TOF and NMR.These compounds include:1-(4-Hydroxyphenyl)-1,2,3propanetriol(1),guaiacylglycerol(2),N5-(4’-hydroxybenzyl)-L-glutamine(3),(E)-pcoumaryl alcohol(4),coniferyl alcohol(5),phenylalanine(6),uracil(7)and thymine(8).Compounds 1-5 were isolated from the genus Cucumis for the first time.4.The HPLC-ELSD method was developed to determine the content of fructose,galactose,sucrose,and gossytose in melo semen extracts and Cervus and Cucumis Polypeptide Injection.The total contents of four kinds of sugars in three different batches of melo semen extracts and Cervus and Cucumis Polypeptide Injection were 145.1μg·mg-1,255.8μg·mg-1,163.lμg·mg-1,and 153.7μg·mg-1,respectively.The contents of reducing sugars measured by phenol sulfuric acid method was 31.4%,34.9%,32.1%,and 19.6%,respectively.The contents of polysaccharide in melo semen extracts was 51.6%,53.6%,52.9%,respectively,which were determined by gradient precipitation.The free amino acids in three different batches of melo semen extracts and deer bone extracts and one batch of Cervus and Cucumis Polypeptide Injection were 6.4μg·mg-1,20.1μg·mg-1,10.1μg·mg-1,51.3μg·mg-1,44.1μg·mg-1,52.4μg·mg-1,and 35.7μg·mg-1,respectively.The total amino acids were determined as 131.7μg·mg-1,138.4μg·mg-1,113.3μg·mg-1,694.4μg·mg-1,724.5μg·mg-1,747.0μg·mg-1and 363.2μg·mg-1,respectively.