Studies On The Active Chemical Compositions Of Panax Quinquefolium. L And Their Fingerprint

Panax quinquefolium.L is a herbal medicine used to decrease stress and reduce high blood sugar. Other names for Panax quinquefolium.L include: American Ginseng, Canadian ginseng, Redberry, Guangdong ginseng and Strawberry ginseng. Ginseng root has been used as a form of tonic medicine in China for more than two thousand years. Panax quinquefolium.L is the North American variety of ginseng native to the United States and Canada. In recent years, pharmacology studies of Panax quenquifolium.L and its compound-prescription for immunity adjusting have developed greatly. Panax quenquifolium.L and its extractions have been employed widely in functional food, medicine and food additive industries. The pharmacology studies showed that the bioactive component in Panax quenquifolium.L were saponins, polysaccharide, et al. Until now, the bioactive components in Panax quenquifolium.L have not been studied profoundly, which could affect the integrated exploitation of Panax quenquifolium.L and its bioactive components.The extraction and determination of polysaccharide in Panax quinquefolium.L were studied in detail in this paper, which will provide foundations for their further developments as matrix of functional food, or as new medicine. A convenient, modern analysis method for the determination of seven major ginsenosides in Panax quinquefolium.L was developed, and different extracting methods of ginsenosides were compared according to the extraction yeilds of seven ginsenosides. ICP/MS and HPLC-ICP/MS were used to study contents of inorganic elements and the elemental speciation in Panax quinquefolium.L for the first time. The classification method was developed systematically for quality assessment of Panax quinquefolium.L with fingerprints and chemometrics. The main studies and results in the paper are mentioned as following:1. Kinds of new extraction technologies on plant active components were applied in extracting of Panax quinquefolium.L polysaccharide. Combined enzymes-reflux extraction was the most effective method by comparing several different extractionmethods (reflux, microwave, supersonic wave, microwave- reflux, supersonic wave-reflux, combined enzymes-reflux, combined enzymes-microwave, combined enzymes- supersonic wave). In order to obtain the optimization parameters range of polysaccharide extracting, enzyme amount, ratio of material to solvent, processing time by enzyme and extracting temperature were inspected as single factor. Based on these, orthogonal experiment was carried out and the optimum technical parameters were obtained: 50 °C of enzymatic reaction temperature for 1.5h, 1:12 of ration of material to solvent, 20 uL.g'1 of enzyme amount, extracts two times at 100 °C.2. An anthrone-H2SO4 colorimetry method was developed for the determination of polysaccharides in Panax quinquefolium.L samples from different origins. Purified polysaccharide in Panax quinquefolium.L, was prepared by extracting with water and removing the interference components such as lipid, monosaccharide, disaccharide, oligosaccharide, amino acids and protein. The optimum absorption wavelength was confirmed by UV-Vis scanning after the reaction of purified polysaccharide and anthrone-H2SC>4. A corrected factor was confirmed by comparing purified polysaccharide and glucose in order to minimize the error of determination. The results showed that the method was simple and available, and the color of the treated samples could be stable within 4 h. The average value of the recovery for the determination of the polysaccharides was 96.6% with 1.33% of RSD (n=4). The contents of polysaccharides in Panax quinquefolium.L from different origins changed from 6.32% to 10.8%.3. A high performance liquid chromatographic method was established for the determination of ginsenoside Rbi in panax quinquefolium.L. Contents of total ginsenoside and ginsenoside Rbi of twelve samples were determined in order to observe the relationship between origin and content. The contents of total ginsenoside were determined by UV-spectrophotometry, and the contents of ginsenoside Rbi were determined by HPLC. The calibration curve was linear over the concentration range of 62~1230 ug.mL'1 for ginsenoside Rbi, the correlation coefficient r was 0.999. The average recovery of the determination of ginsenoside Rbi was 99.2% with the RSD of 1.69% (n=6). The contents of total ginsenoside often samples are more than4%, while contents of of ginsenoside Rbi of ten samples are more than 1%. The developed method was found to be simple, fast, accurate and reliable for the determination of ginsenoside Rbi.4. A convenient, modern analysis method for the determination of seven major ginsenosides in Panax quinquefolium.L was developed to determine the contents of seven ginsenosides of twelve samples and the chromatographic fingerprint of Panax quinquefolium.L ginsenosides was established by high performance liquid chromatography. Separations were carried out by linear gradient elution using a alltima Cig (250mmx4.6mm, 5 um) colum with acetonitrile-0.05% phosphoric acid as mobile phase and detection length as UV203 nm, the flow-rate was 1.2 mL.min"1, the colum temperature was 35 °C . Results indicated that the linearity of seven ginsenosides of this method was well and the average recovery of the determination of seven ginsenosides was 94.1—97.9%. Clustering analysis and the similarity of the HPLC chromatogram was performed for classification of samples, which obtained a good result. This method provides useful information for quality control of traditional Chinese medicine, and is a development for the application of HPLC in sample analysis and identification of complex components.5. Different extracting solvent and method of ginsenosides were compared according to the extraction yeilds of seven ginsenosides. Results showed that the extraction ratio of seven ginsenosides with different solvent and methods was different. Theoretic and experimental basis were offered reliably for the optimization of ginsenosides extraction by this results, which could establish the basis of mono-ginsenoside separation and purification in Panax quinquefolium.L6. ICP/MS and HPLC-ICP/MS were used to study contents of inorganic elements and the elemental speciation in Panax quinquefolium.L for the first time. Chloroform, water and ethyl alcohol were employed to extract several elements in Panax quinquefolium.L, which were determined by inductively coupled plasma mass spectrometry (ICP-MS) in order to analyze the transference characteristics of elements. The coupling of high performance liquid chromatography (HPLC) with inductively coupled plasma mass spectrometry (ICP-MS) was applied for the analysisof elemental speciation. Results showed the polarity of extraction solvent could highly effects the solvent possibility of the analyzed element. The transference characteristics of kinds of elements including Mg, Ca, P, Mn, Sr, Mo, Pb were in linearity proportion with the polarity of extraction solvent. The inorganic element speciation and the organic element speciation were separated by HPLC-ICP/MS on the basis of C-18 bonded silica gel column with 0.10 mol.L"1 nitric acid as mobile phase at flow rate of 0.4 mL.min"1, injection: 20 \xL. The determination results of inorganic elements in Panax quinquefolium.L extraction showed that the contents of inorganic elements in polysaccharide were more than in Panax quinquefolium.L and the inorganic elements contents in different polysaccharide which was extracted by different methods were discrepant. The contents of Cr, Ni, Cu, Zn, Pb in total ginsenosides of Panax quinquefolium.L were higher than in Panax quinquefolium.L and other elements were lower. The theoretic basis was offered for the sufficient exploitation and pharmic study of polysaccharide and total ginsenosides.7. The inorganic elements fingerprint of Panax quinquefolium.L was developed for the first time. The contents of elements such as Mg, Al, P, Ca, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Sr, Mo, Cd, Pb in twelve Panax quinquefolium.L samples were determined by ICP/MS. The results were used for the development of element chromatogram. The principal component analysis of SPSS was applied for the study of characteristic elements in Panax quinquefolium.L. Five principal components were found from original data which showed that Fe, Al, V, Mn, Mg, Sr, Mo, Ca, Cu could be the characteristic elements in Panax quinquefolium.L. The cluster analysis was used for the classification of Panax quinquefolium.L. The results of Q-type cluster analysis showed that the samples could be clustered reasonably into five groups and the elemental distribution characteristics were related to the breeds of Panax quinquefolium.L. It was found that the principal component analysis and cluster analysis were useful in element analysis of Panax quinquefolium.L.8. The chemometrics method was developed firstly for classification of Panax quinquefolium.L. The pattern recognition based on principal component analysis (PCA) and cluster analysis was applied to the measured data (23 variables) of thePanax quinquefolium.L samples. The results showed that twelve samples were classified reasonably by principal component and the contents of seven ginsenosides were the first key factor for classification, the contents of Mn, Cu, As, Ni, Mo elements and polysaccharide were the second key factor. The analysis results showed that the classified results changed with differentia of cluster analysis variables and it was reasonable that the cluster analysis of Panax quinquefolium.L. was performed by integrated analysis variables of organic components and inorganic elements. The principal component analysis and cluster analysis were useful in classification of Panax quinquefolium.L.