Studies On Chemical Constituents In The Leaves Of Panax Ginseng C.A. Meyer And Their Bioactivities

Panax ginseng C. A. Meyer (P. ginseng) as a plant of Araliaceae is mainly distributed in Northeast region and Changbai Mountain of China, North Korea, Corea, Japan and Russia. As early as the 1970′s, the aboveground parts of P. ginseng have been studied by many scholars from different countries, who found that the leaves of P.ginseng contains the same ginsenosides with the roots, and the content was higher than the roots. The leaves of P.ginseng have been widely used throughout the word for the treatment of a number of diseases, including anaemia, diabetes, cancer and arrhythmia etc.The main chemical constituents are ginsenosides which are glycosides that contain an aglycone with dammarane skeleton in the leaves of P.ginseng. From 1976, there have been more than forty saponins in the leaves of P.ginseng. In order to further study on the chemical constituents and bioactivities of the leaves of P.ginseng, the extraction, separation and determination of the leaves of P.ginseng from Jingyu country of Jilin Province were preformed. 10 saponins (GinsenosideF2, GinsenosideRd, GinsenosideF5, GinsenosideRb3, GinsenosideRb1, GinsenosideRb2, GinsenosideRc, GinsenosideF1, GinsenosideF3 and Notoginseoside Fe) in P. ginseng leaf were simultaneously determined by RP-HPLC, and the contents of total saponins in P. ginseng leaf from different areas in China were compared. In addition, the contents of 10 saponins in Huoliyuan tablets which were traditional chinese medicine were also quantitatively determined.13 saponins (GinsenosideRb1, GinsenosideRb2, GinsenosideRb3, GinsenosideRc, GinsenosideRd, GinsenosideF1, GinsenosideF2, GinsenosideF3, GinsenosideF5, Notoginseoside Fe, Protopanaxadiol, GinsenosideRg2, Majonoside F4), b mixed compounds (GinsenosideRb1, GinsenosideRb2, GinsenosideRb3, GinsenosideRc, GinsenosideRd) and F mixed compounds (GinsenosideF1, GinsenosideF2, GinsenosideF3, GinsenosideF5, Notoginseoside Fe, GinsenosideRg2) were studied by inhibiting AAPH (2,2′-azobis (2-amidinopropane )hydrochloride) induced hemolysis of human erythrocytes in thesis.The air-dried leaves (2kg) were extracted three times with water under heating. The extracts were combined, subjected to a macroporous absorption resin AB-8 column by eluting with water to remove impurities, and then eluted with 85% EtOH. The eluate was collected and evaporated under vacuum to afford the crude saponins fraction, which was partitioned in an EtOAc-H2O (1:1 v/v) mixture. The aqueous phase was further extracted with n-BuOH. Removal of the solvent from the EtOAc-soluble, n-BuOH-soluble and H2O-soluble fractions under reduced pressure yielded 91g, 106g, 33g of the residue, respectively. The compounds were separated and purified by silica gel column, ODS column, RP-HPLC etc.. Twenty-four compounds were isolated and identified by 1DNMR,2DNMR,MS etc., as compound 1(Ginsenoside A),compound 2(Ginsenoside B),compound 3(Ginsenoside C),compound 4(Ginsenoside M7cd),compound 5(Ginsenoside Rg6),compound 6 (20-(E)-Ginsenoside F4),compound 7(Gypenoside XVII),compound 8(Ginsenoside Rb3),compound 9(Gypenoside IX),compound 10(Majoroside F4),compound 11 (Ginsenoside F1),compound 12(Ginsenoside F2),compound 13(Ginsenoside F3),compound 14(Ginsenoside F5),compound 15(Ginsenoside Rd2),compound 16(Notoginseoside Fe),compound 17( 20-(S)-Ginsenoside Rh1 ),compound 18 ( Ginsenoside Re ),compound 19(20-(R)-Ginsenoside Rg2),compound 20(20-(S)-Ginsenoside Rg2),compound 21(Ginsenoside Rb1),compound 22(Ginsenoside Rb2),compound 23(Ginsenoside Rc),compound 24(Ginsenoside Rd). Among which, compond 1-9 are the first time isolated from the leaves of P.ginseng, compound 1, 2, 3 are new , named ginsenoside A, B, C.In this thesis, The conditions of HPLC-UV were firstly established for the quantitative and qualitative determination of 10 saponins(GinsenosideF2, GinsenosideRd, GinsenosideF5, GinsenosideRb3, GinsenosideRb1, GinsenosideRb2, GinsenosideRc, GinsenosideF1, GinsenosideF3 and Notoginseoside Fe) in P. ginseng leaf and Huoliyuna tablets. The appropriate analytical condition: UV absorption was measured at 203nm.Gradient elution was employed using solvent A (acetonitrile) and solvent B (water) at 25℃, the gradient eluting program started with 29.6% (v/v) solvent A and 70.4% (v/v) solvent B for 15 min, then 15-30min, changed to 40% (v/v) solvent A and solvent B 60% (v/v), and was held for 10 min. The flow rate was kept at 1 ml/min and the sample injection volume was 10μL. The results showed that the content of ginsenosideRd was the highest, the contents of ginsenosideRb1, Rb3, notoginseoside Fe were lower than the other ginsenosides. the contents of the saponins were obviously different from different growing area. the contents of total saponins in the leaves from changbai were lower than the leaves from other growing area. the contents of ginsenoside Rb3 in the leaf of P. ginseng cultivated in Fusong was higher than the other samples.The quantification method is rapid, accurate, and precise. The proposed method satisfies the need for quality control of P. ginseng leaves and their products.13 saponins, F mixed compounds and b mixed compounds were studied by inhibiting AAPH (2,2′-azobis(2-amidinopropane)hydrochloride) induced hemolysis of human erythrocytes.The results showed that a majority of ginsenosides obviously protect human erythrocytes against AAPH-induced haemolysis. It showed that the effects changed duing to different compounds, and the time when hemolysis attained 50% was also different. Ginsenoside F2 , Rd , Rg2 , F5 , Rb3 and ppd exhibited more protevtive effect, but ginsenoside F1, F3, F5 and notoginsenoside Fe exhibited little protevtive effect.The results showed that the free-radical-scavenging activity of various ginsenosides relate to the type of ginsenoside, the category, the number, the position and the connect of the the sugar moieties. In addition, the mixed ginsenosides played the most efficient protective role, which was possibly caused by the synergistic reaction among several compounds and was expected for the further continued research. The active sequence was F mixed compounds> b mixed compounds > F2 > ppd > Rd > Rg2 >F5 > Rb3 > Rb1 > N-Fe > MF4 > Rb2 > Rc > F1~F3. The presented result provides useful information for the pharmacological research of ginsenosides.