| Panax japonicus C. A. Mey. var. major (Burk.) C.Y.Wu et K.M.Feng, being listed among Eight KingKong of Taibai qiyao, is one of the main Chinese medicinal plants. There are two varieties of Panax(P. japonicus C. A. Mey. var. major (Burk.) C.Y.Wu et K.M.Feng and P. japonicus C. A. Mey. var. bipinnatifidus (Seem.) C.Y.Wu et K.M.Feng) recorded as the original plants in the Chinese Pharmacopoeia. Popular as it is in folk medicine, yet the application and exploitation of P. japonicus C. A. Mey. var. major are far from its due. The reason mainly lies in it that we lack in the studies on chemical substances and screening of the active components. In this study, total saponins, free fatty acids(FFAs) in ethanol extract, polysaccharide and its monosaccharide composition, liposoluble constituents in different parts from P. japonicus C. A. Mey. var. major were quantitative analyzed, and antioxidant and antibacterial activities of several extracts were studied. The screening model for active compounds was established and applied to liposoluble constituents of alcoholic extract from P. japonicus C.A. Mey. var. major. The study would provide scientific information for the rational drug administration and further utilization.The main contents of this dissertation are as follows:1. Comparison between the extraction yields of total saponins with different methods from the rhizome of P. japonicus C. A. Mey. var. major. The best technological conditions for the ultrasonic-assisted extraction were obtained:ultrasonic time, ultrasonic temperature, ethanol concentration and ratio of gardenia to liquor were 30 min,50℃,50% and 1:20, respectively. The extraction yields obtained with ultrasonic-assisted extraction (8.30%) was considerably higher than those obtained with immersion (5.06%) and reflux method (6.30%)2. Simultaneous determination of ginsenoside R,Rf and Rb1 in the rhizome of P. japonicus C.A. Mey. var. major. RP-HPLC Simultaneous determination of ginsenoside Re,Rf and Rb1 was established, optimized experimental conditions were as follows: mobile phase was CH3CN (A)-H2O (B), the gradient elution was programmed 0-35 min,19%A; 35-55 min,19%A-29%A; 55-65 min,29%A; mobile phase was set at a flow rate of 0.8 mL/min; injection volume was set 10μL; the detection wavelength was set 203 nm. The method is sensitive, accurate and reliable. The method has been applied to the determination of ginsenoside Re,Rf and Rb1 from the rhizome of P. japonicus C. A. Mey. var. major. Experiment results revealed that the medicinal plant was abundant in ginsenoside, the percentage mass content of Re,Rf and Rb1 were 0.072%,1.341% and 0.064%, respectively.3. Study on optimization of the ultrasonic extraction conditions of total phenolic acids in rhizome of P. japonicus C. A. Mey. var. major, The best technological conditions were obtained by orthogonal experiment design. They were 60℃,30 min, ethanol concentration 50% and a ratio of gardenia to liquor of 1:20, respectively. The extraction yield of total phenolic acids under the optimized conditions was 0.273%.4. FFAs profile analysis in alcohol extract of rhizome of P. japonicus C. A. Mey. var. major. The method for the methyl esterification of FFAs was optimized, the quantitative method for the determination of FFAs by GC-MS was established and applied to the analysis of FFAs in alcohol extract of P. japonicus C. A. Mey. var. major and P. japonicus C.A.Mey. var. bipinnatifidus. Six main FFAs were identified in the alcohol extract of both medicinal plants; they are palmitoleic acid, palmitic acid, linoleic acid, linolenic acid, oleic acid and stearic acid. The content of FFAs in the alcohol extract from P. japonicus C. A. Mey. var. major is 0.53mg/g,4.74 mg/g,2.31 mg/g,1.02 mg,0.12mg/g,1.54 mg/g, respectively, The content of FFAs in the alcohol extract from P. japonicus C.A.Mey. var. bipinnatifidus 0.12mg/g,0.98mg/g,0.54mg/g,0.25mg/g,0.30mg/g,0.20mg/g, respectively.5. Study on the composition of monoses hydrolyzied from polysaccharide of the rhizome of P. japonicus C. A. Mey. var. major. The hydrolysis method for polysaccharide and the derivative method for monosaccharide were optimized. A GC-MS method was developed for identifying and quantifying(normalization method) the hydrolyzied monoses. The results revealed that 7 monoses were found hydrolyzied from polysaccharide of P. japonicus C. A. Mey. var. major, they were rhamnose (1.88%),ribose (5.22%), arabinose (2.21%), xylopyranose (3.59%), mannose (4.08%), glucose (78.13%), galactose (4.21%)6. The liposoluble constituents from different parts of P. japonicus C. A. Mey. var. major. The results revealed that components from rhizome were quite different from those from leaves. Compounds composition analysis provided favorable evidence for the regularity of prescription about this medicinal plant, in which many pharmacologically valuable components with high content were found in different parts, such as hexadecanoic acid, linoleic acid, stearic acid, falcarinol,β-sitosterol, squalene, and so on. The results revealed that the liposoluble components from P. japonicus C. A. Mey. var. major did have high pharmaceutical value. 7. Study on the antioxidant activities of different extract from the rhizome of P. japonicus C. A. Mey. var. major. The antioxidant activities were evaluated with DPPH free radical scavenging assay andβ-carotene-linoleic acid assay. The results showed all the extracts tested all possessed antioxidant capacity, and the acetone extracts showed higher antioxidant activity than other extracts. The order of the capacity to scavenge DPPH free radical was in accord with that of total antioxidant capacity, ie. acetone extracts>methanol extracts>total saponins.8. Study on the antibacterial activities of different extracts from the rhizome of P. japonicus C. A. Mey. var. major. All extracts from P. japonicus C.A.Mey. var. major had showed different degrees of antibacterial activity on pathogenic bacteria tested, including Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus white, Bacillus subtilis, Bacillus coagulans and Bacillus cereus. The total saponins showed better bacterial activities.9. At the advantage of GC-MS techniques and special affinity for active receptors on CMCM, we established a drug discovery model for screening active compounds for cardiovascular diseases. An online method for determining ATPase bioactivity was so developed as to indirectly investigate the activities of acceptors on CMCM. experimental results revealed that the bioactivity of ATPase decreased with increasing lapse of time in vitro in buffer solution, while the ATPase on CMCM remained bioactive over a certain time (at least 9 h). It reflected the rule of the variation of the acceptor activities, which indicated that CMCM was eligible for pertinent medicine discovery. The screening model was applied to the liposoluble components of alcoholic extract of P. japonicus C.A.Mey. var. major. CMCM at different activity levels was incubated with the liposoluble components of the extract from rhizome of P. japonicus C.A. Mey. var. major, and then the individual liposoluble compounds underwent separation and identification on GC-MS. With probing the variation of the content of the identified compounds before and after the incubation, we preliminarily screened the potentially active compounds.Experimental results revealed that under the developed discovery model two compounds in the liposoluble components of the extract were found to have shown specific affinity, they wereβ-sitosterol and another unidentified compound.The experimental results laid the foundation for the further perfection of drug discovery model in molecular level.
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