| 1. The extraction of ginsenosidesFor the purpose of preparing anti cancer active components-20(S)-protopananxadiol and 20(S)-ginsenoside Rh2, high content protopananxadiolsaponins must be contained in the total ginsenosides. So the suitable plantmaterials of Ginseng should be selected before the total ginsenosides wereextracted. By HPLC determination, the total content of protopananxadiolsaponins and protopanaxatriol saponins in the leaves of Quinquefolium L. were3.57% and 0.87% respectively (the ratio is 4.1:1), while they were 0.19% and0.09% respectively in the stems of the plant (the ratio is 2.1:1). Moreover, thereare more amount of total ginsenosides in the leaves than in the stems ofQuinquefolium L. ( the ratio is 15.8:1). Thus, the leaves of Quinquefolium L. isthe optimal plant material for obtaining ginsenosides.The AB-8 macroprous adsorption resin was used to extract theginsenosides from the leaves of Quinquefolium L. The influencing factors onthe extraction rate of ginsenosides were tested before optimal extractingcondition were selected and it can be concluded that 25 times water (v/w) wasused in extraction at boiling point for 4 times, and the time were 2.5,2,1.5 and1 h for each times respectively, then the total saponins in the water solutionwere adsorpted by AB-8 microporous resin at a flow rate of 3ml/cm2.min. 85%ethanol was used for eluation and the total saponin were gained after ethanolwas removed by distillation. The protopananxadiol saponins andprotopananxatriol saponins were dedermined to be 49.93% and 10.34%respectively in the extract, and the ratio is 4.8:1. The yield of protopanaxadioland protopanaxatriol ginsenosides are 3.55% and 0.73% respectively. Theextraction rate of the total ginsenosides from the leaves was hgiher than 7%.The results showed that both the extraction rate of ginsenosides and the contentof protopananxadiol saponins were high in the extract.2. Ginsenoside degradationThe strong acid (hydrochloric acid), weak acid (acetic acid), strong alkali(sodium hydroxide) water solution and strong alkali(sodium hydroxide)glycerol solution were used to degrade the total ginsenosides from the leaves ofQuinquefolium L. The optimal conditions in the four medium were found byprimary tests, and the results showed that there was little amount of20(S)-protopananxadiol and 20(S) ginsenoside Rh2 in the degradation productsfrom hydrochloric acid, acetic acid and sodium hydroxide water solution. Thehighest transformation rate in the three methods mentioned above is in thehydrochloric acid solution, but the transformation rate of degradation was justhigher than 10%, but much lower than that from sodium hydroxide glycerolsolution. So, it is difficult to degrade ginsenosides effectively.The optimal condition in sodium hydroxide glycerol solution wasestablished by orthognol experiments. The optimal conditions of degradation toprepare 20(s)-ginsenoside Rh2 are as follows: the total saponins and sodiumhydroxide (the ratio is 1:1.6, saponins/ sodium hydroxide, w/w) are dissolvedin glycerol (the ratio is 1:15.0, w /v), then the solution was heated up to 220℃and was keeped for 40 min at the temperature, the transformation rate was55.64%.The optimal condition of degradation to prepare 20(s)-protopanaxadiolare as follows: the total saponins and sodium hydroxide (the ratio is 1:2.0, w/w)are dissolved in glycerol (the ratio is 1:15.0, w/v) , then the solution was heatedup to 235℃ and was keeped for 200 min at the temperature , thetransformation rate was 85.93%. The results showed that ginsenosides can bedegraded and become minor ginsenoside with one sugar in carbon 3 or becomesapogenin by the two degradation methods above mentioned.3. The isolation and identification of ginsenosides degradation productsThe chromatography and recrystallization were used to isolate theconstituents from the degradation products coming from sodium hydroxideglycerol solution, hydrochloric acid solution and acetic acid solutionrespectively. All the compounds were identified by the spectral methodsincluding 1H-NMR,13C-NMR ,MS,UV and IR etc.Ten compounds were identified from the two degradation products ofsodium hydroxide glycerol solution as 20(S)-ginsenoside Rg3, 20(S)-ginsenoside Rh1, 20(S)-ginsenoside Rh2, 20(S) -ginsenoside Rh3, 20(S)-protopananxadiol, 25-ene-20(S)-protopananxadiol, 20(S)-protopananxatriol,20(S), 24(R)-epoxy-protopanaxatriol, 20(S)-24-methyl -23–ene–24–carbonyl-pratopanaxadiol and 25-hydroxyl -23-ene-20(S)-protopananxadiol. Among them, 25 -ene-20(S) protopananxadiol , 20(S)-24-methyl -23 – ene – 24 – carbonyl -pratopanaxadiol and 25 -hydroxy-23-ene-20 (S)-protopananxadiol are found for the first time from the ginsenosidealkali degradation products, and 20(S)-24-methyl -23 –ene–24–carbonyl -pratopanaxadiol and 25-hydroxy-23-ene-20 (S)-protopananxadiol are newcompounds according to the literature.Two compounds were identified respectively from degradation productsof sodium hydrochloric acid solution and acetic acid solution, and they are20(R)-panaxadiol and 20(R)-ginsenoside Rg3.According to the analysis on the compounds isolated from degradationproducts, and the reaction mechanism, the by-products and complexconstituents can be found in the degradation products from hydrochloric acidsolution , some ginsenosides can transform into 20(S)-panaxadiol or 20(S)-panaxatriol, meanwhile, some ginsenosides transform into 20(S)-and 20(R)-sapogenins. In the acetic acid solution, 20 position carbon of ginsenosides canbe transform from S to R isomer, and the degradation reaction process isuncomplet. In the alkali glycerol solution at high temperature, the ginsenosidescan be degraded into minor saponin or sapogenin with its 20-S isomerunchangeable and degradation reaction process is complete.4. The quantitative analysis of the main constituents from degradationproductsThe optimal HPLC conditions were established for 20 (S)-protopananxadioland 20 (S)-ginsenoside Rh2 determination after the method had been tested. Ateach dedermination condition, 203nm is the maximal adsorption wavelengthfor the two compounds. The methanol and water were mixtured as mobilephase with the flow rate of 1.2ml/min and the ratio were 90:10 and 85:15 for20 (S)-protopananxadiol and 20(S)-ginsenoside Rh2 respectively. The detectionlimits are 0.05 μg and 0.025μg , the RSDs are 0.74% and 0.72%, thereproducibilities are 0.37% and 0.27%, and the recoveries are 100.6% ( RSD=1.80% ) and 100.2% (RSD=0.91%) for 20(S)-protopananxadiol and 20(S)-ginsenoside Rh2 ,respectively, and the linear range were 0.5-20 μg for thetwo compounds with the correlation coefficient of 0.9999.The purities of 20(S)-protopananxadiol and 20 (S) ginsenoside Rh2 wereincreased greatly by column chromatography and recrystallization and reachedto 97.7% and 92.9% respectively. Moreover,the average content of 20 (S)-protopananxadiol and 20(S)-ginsenoside Rh2 in their degradation products are25.4% and 12.7%,respectively.5. Anti tumor effect of the main degradation productsThe anti tumor effect in vivo tests showed that 20(S)-protopananxadiol caninhibit mice liver cancer H22,Lewis lung cancer and melanoma B16 in adose-dependent manner. At 50mg/Kg and 100mg/Kg, the average rates ofinhibitory mice liver cancer H22 were 44.11% and 48.56%, the average rates ofinhibitory mice Lewis lung cancer were 48.58% and 48.67%, the average ratesof inhibitory mice melanoma B16 were 39.27% and 40.80% respectively.However, the inhibitory rate was low in lower dose group (25mg/ml).The anti tumor effect in vitra tests showed that 20(S)-ginsenoside Rh2 canreduce Hep-A-22 cells survival ability and induce apoptosis in dose-dependentmanner. When the concentration of 20(S)-ginsenoside Rh2 was 100μg/ml, thecells viability decreased to lower than 10%, the apoptosis rate was 60%, thecycle distribution of cells in G1 phase was prolonged and enhanced the expressionof Bax in cytoplasma.In conclusion,all the results of the study can offer credible foundation forthe development of new anti tumor drugs.
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