Study Of Common Features Of Lingzhihuang Capsule And Its Formula Medicine Of Acidic Hydrolysis Polysaccharides Based On The HPLC Fingerprint Technology

Traditional Chinese medicine polysaccharide is a macromolecular substance,which has immunomodulatory activity and antitumor effect.It' s used Phenol-sulfuric acid colorimetric method to determine the content of total polysaccharides,and the research on fingerprint is less.Traditional Chinese medicine fingerprint technology is recognized as the most effective method of traditional Chinese medicine quality control at home and abroad.This study choose the chinese medicine compound of Lingzhihuang Capsule(a kind of health care product extracted according to certain proportion with kinds of Traditional Chinese medicine containing of Ganoderma lucidum,Polygonatum,Lycium barbarum,Radix Ophiopogonis,atractylodes rhizone,Indian bread and Schisandra chinensis extracted)and single Chinese medicine of Ganoderma lucidum,Polygonatum,Lycium barbarum and Radix Ophiopogonis containing polysaccharide as the research object,using the traditional Chinese medicine fingerprint technology,to establish the polysaccharide of acid hydrolysis of fingerprint method and identify of the polysaccharide structure.Objective1.To discuss the establishment acidic hydrolysis polysaccharides of Lingzhihuang Capsules and formula medicine Ganoderma lucidum,Polygonatum,Lycium barbarum and Radix Ophiopogonis HPLC characteristic spectrum analysis method is feasible.2.To screen Lingzhihuang Capsules and formula medicine Ganoderma lucidum,Polygonatum,Lycium barbarum and Radix Ophiopogonis of the preparation of test sample solution method,and evaluate the stability of polysaccharide.3.To compare the establishment mode of Ganoderma lucidum,Polygonatum,Lycium barbarum and Radix Ophiopogonis of HPLC characteristic spectrum commonalities and differences.Methods1.Using the HPLC characteristic spectrum analysis method of Lingzhihuang Capsules and formula medicine Ganoderma lucidum,Polygonatum,Lycium barbarum and Radix Ophiopogonis of polysaccharides to analysis 10 batches of samples,specific chromatogram similarity evaluation system software writed by the State Pharmacopoeia Commission Specific chromatogram similarity evaluation system software(2004A version)was adopted to generate a total of mode.2.Filtering samples of polysaccharide extraction method,in trifluoroacetic acid(TFA)0.4 mol/L,with hydrolysis temperature 110 ?,in 1-phenyl-3-methyl-5-pyrazolone(PMP)0.5 mol/L,with temperature 70 ?,it was determined the best hydrolysis time and derivativing time.3.It was established acidic hydrolysis polysaccharides of Lingzhihuang Capsules,Ganoderma lucidum,Polygonatum,Lycium barbarum and Radix Ophiopogonis HPLC characteristic spectrum analysis method,to generate a total of mode,and to evaluate.the similarity,and to discuss the characteristics of Lingzhihuang Capsules peak and single medicine belongs to.4.Based on the relative retention time qualitative of monosaccharide,it's identify respectively monosaccharide of composition of polysaccharides with the above samples.5.To compare the establishment mode of Ganoderma lucidum,Polygonatum,Lycium barbarum and Radix Ophiopogonis of HPLC characteristic spectrum commonalities and differences.Results1?The establishment of the composition of polysaccharides qualitatirve identification and fingerprint was through removing liposoluble components,extracted with aqueous,alcohol precipitation,acidic hydrolysis and derivation.(1)It,s determined the polysaccharide solution concentration of Lingzhihuang Capsules was 0.18g/mL and the best alcohol concentration was 80%with sample quantity for 10 u L.The optimum hydrolysis and derivative time of the Lingzhihuang capsule polysaccharide solution were determined for 6 h and 60 min.(2)The analysis method accorded with the requirements of the Chinese traditional medicine fingerprint technology.10 batches of samples were marked out of a total of 14 common peaks,and samples' similarity were 0.976~0.997.The eight characteristic peak were identified,including D-Mannose,L-Rhamnose,D-galacturonic acid,D-Glucose,D-Galactose,D-Xylose,D-arabinose and L-mycose etc.(3)Peak 10(D-Glucose)was the strongest peak,more than 85.0%of total peak area,belonging to Ganoderma lucidum,Radix Ophiopogonis,atractylodes rhizone,Poria cocos.Peak 11(D-Galactose)belongs to Ganoderma lucidum,Polygonatum,Radix Ophiopogonis,Poria cocos.Peak 1(D-Mannose)belongs to Radix Ophiopogonis,Poria cocos,Schisandra chinensis.Peak 14(L-Fucose)belongs to Ganoderma lucidum,atractylodes rhizone,Poria cocos.2.Using the HPLC characteristic spectrum,it' s established HPLC characteristic spectrum analysis method of Ganoderma lucidum,Polygonatum,Lycium barbarum and Radix Ophiopogonis.The analysis method accorded with the requirements of the Chinese traditional medicine fingerprint technology.(1)With common peak area of indicators,it' s determined the polysaccharide solution concentration of Ganoderma lucidum,Polygonatum,Lycium barbarum and Radix Ophiopogonis was respectly 0.4 g/mL?0.5 g/mL?0.5 g/mL?.0.6 g/mL and the best alcohol concentration was 80%with sample quantity for 30 ? L?20 ?L?30 ? L?30 ? L.The optimum hydrolysis and derivative time of the polysaccharide solution were determined for 6 h?7h?6h?6h and 100 min?40 min?60 min?60 min min.10 batches of Ganoderma lucidum samples were marked out of a total of 12 common peaks,and samples' similarity were 0.973-0.992.The seven characteristic peak were identified,including D-Mannose,L-Rhamnose,D-glucuronic acid,D-Glucose,D-Galactose,D-Xylose and L-mycose etc.D-Glucose was the strongest peak,more than 70.0%of total peak area,followed by D-Galactose,D-Mannose and L-mycose.(3)10 batches of Polygonatum samples were marked out of a total of 10 common peaks,and samples' similarity were more 0.99.The seven characteristic peak were identified,including D-Mannose,L-Rhamnose,D-galacturonic acid,D-Glucose,D-Galactose,D-Xylose and D-arabinose etc.D-Galactose was the strongest peak,more than 55.0%of total peak area,followed by D-galacturonic acid,D-Glucose and D-Mannose.(4)10 batches of Lycium barbarum samples were marked out of a total of 12 common peaks,and samples' similarity were more 0.99.The eight characteristic peak were identified,including D-Mannose,L-Rhamnose,D-glucuronic acid,D-galacturonic acid,D-Glucose,D-Galactose,D-Xylose and D-arabinose etc.D-arabinose was the strongest peak,more than 45.0%of total peak area,followed by D-Galactose,D-Xylose and D-galacturonic acid.(5)10 batches of Radix Ophiopogonis samples were marked out of a total of 9 common peaks,and samples' similarity were more 0.98.The eight characteristic peak were identified,including D-Mannose,L-Rhamnose,D-galacturonic acid,D-Glucose,D-Galactose,D-Xylose,D-arabinose and L-mycose etc.D-Galactose was the strongest peak,more than 50.0%of total peak area,followed by D-Mannose,D-Glucose and D-arabinose.(6)Comparing four kinds of medical model similarity,the similarity ratio is 0.187,0.126,0.622 with Ganoderma lucidum polysaccharides compared with Polygonatum,Lycium barbarum and Radix Ophiopogonis.The similarity ratio is 0.496.0.473 with Polygonatum compared with Lycium barbarum and Radix Ophiopogonis.The similarity ratio is 0.540 with Lycium barbarum compared with Radix Ophiopogonis.3.Analysis of common features of acidic hydrolysis of polysaccharides in Ganoderma lucidum,Polygonatum,Lycium barbarum and Radix Ophiopogonis was based on the HPLC fingerprint technology.(1)The common features of preparation of test sample solution.? Using ultrasonic extraction by two times with 80%ethanol to remove liposoluble components.? Polysaccharide can be extracted completely with water by 1-2 times.The solution of crude polysaccharide was 0.4g/mL-0.6g/mL and test sample solution was 0.025 g/mL-0.06 g/mL.Sample quantity for 10-30 ?L are feasible? The optimal hydrolysis time of the four medicine was 6-7 h and better derivative time was 40-60min.(2)Common features and differences of Lingzhihuang Capsule and its formula medicine of acidic hydrolysis polysaccharides.Polysaccharide includes D-Mannose,L-Rhamnose,D-galacturonic acid,D-glucuronic acid,D-Glucose,D-Galactose,D-Xylose,D-arabinose and L-mycose etc.Monosaccharide ratio is large difference between different herbs.(3)Polysaccharide acid hydrolysis column before derivative-HPLC fingerprint method of Ganoderma lucidum,Polygonatum,Lycium barbarum and Radix Ophiopogonis was feasible to identify medicinal materials.Comparison through four kinds of medicinal materials of model similarity,similarity of Ganoderma lucidum polysaccharide compared with Polygonatum,Lycium barbarum and Radix Ophiopogonis were 0.187,0.126,0.622,respectively;Similarity of Polygonatum compared with Lycium barbarum and Radix Ophiopogonis were 0.496?0.473;Similarity of Lycium barbarum compared with Radix Ophiopogonis was 0.540.Similarity between different medicinal materials is very low and the method is to identify herbs.Conclusion(1)The results show that the method of acidic hydrolysis polysaccharides based on the HPLC fingerprint technology is scientific and reasonable.(2)Through the similarity evaluation and identification of characteristic peak,the study masters the composition of polysaccharides of the above traditional Chinese medicine compound and traditional Chinese medicine.(3)Fingerprint similarity degree is very low in different herbs polysaccharides acid hydrolysis,which can be used to identify different medicinal materials.(4)Because the ratio of monosaccharide composition is different among different medicinal herbs,the results of the study can provide effective experimental basis for the quality control of traditional Chinese medicine compound and traditional Chinese medicine,and provides an experimental basis for other traditional Chinese medicine.