Study On The Preparation Method Of Protopanaxadiol Type Minor Ginsenosides

Ginsenosides, as the main functional components in the plants of panax, possess various pharmacological activities. Ginsenosides are triterpenoid saponins which consisting of two parts, the sapogenins(tetracyclic or pentacyclic triterpenoid) and glycosyls. The minor ginsenosides are the degration products when partial sugar have been hydrolyzed and lost from the main ginsenosides. They can be conversed to the sapogenins after all of the sugar degraded.The protopanaxadiol-type ginsenosides are reported that have many pharmacological activities, such as immune enhancement, antifatigue, antineoplastic, anti-hypoxia, anti-aging and anti-hyperglycemic etc. Lots of researches indicated that the protopanaxadiol-type ginsenosides have higher pharmacological effects on antineoplastic than potopanaxatriol-type ginsenosides, and their pharmacological activity is also affected by the number of connected sugar groups. The pharmacological activity of the single glycosides is the strongest and the activity of the two glycosides and the three glycosides decrease in turn.Up to now, among the protopanaxadiol-type secondary ginsenosides, 20(R)-ginsenoside Rg3 and 20(S)-ginsenoside Rh2 have relatively perfect preparation method, but there is no effective large scale production method of 20(R)-Rh2 ginsenosides and 20(S)-ginseng-Rg3.The purpose of this study is to find preparation method of 20(R)-ginsenoside Rh2 and 20(S)-ginsenoside Rg3 and then to provide monomer compounds for the study of its biological activity and the comparative biological activity of R/S isomer.First, two kinds of degradation products of acid and alkali were prepared by the method of literature, with the total saponins from Panax notoginseng stalks and leaves as raw material. Four kinds of compound were isolated from degradation products by the means of silica gel, ODS column chromatography, recrystallization and other isolational methods. They were identified as 20(S)-ginsenoside Rh2, 20(R)-ginsenoside Rh2, 20(S)-ginsenoside Rg3 and 20(R)-ginsenoside Rg3 by the analyses of their physico-chemical properties and NMR spectra data. The standard product of the target was provided for the following experimental contents, such as the preparation process and the content determination.Base on the above mentioned research, the further study determined the best degradation condition of the preparation of ginsenoside 20(R)- ginsenoside Rh2 and 20(S)-ginsenoside Rg3 respectively with the total saponins from Panax notoginseng stalks and leaves as raw material, by single factor test coupling orthogonal test design. The results are as follow:The best degradation condition of the preparation of 20(R)-ginsenoside Rh2: the concentration of HAc(V/V,m L/m L) is 20%; the soild-liquid(m/V,g/m L)is 1:50; 90℃;The degradation time is 1.5h. Using the best degradation condition of the preparation, the yield is 10.7%, according to protopanaxadiol type saponin from Panax notoginseng stalks and leaves( Rb1,Rb2,Rb3,Rc and Rd). The best degradation condition of the preparation of 20(S)-ginsenoside Rg3:the soild-liquid(m/V,g/m L) is 15:1; the concentration of Na OH(V/V,g/m L)is 16%; 210℃; the degradation time is 40min;Using the best degradation condition of the preparation, the yield is 34.80%, according to protopanaxadiol type saponin from Panax notoginseng stalks and leaves(Rb1,Rb2,Rb3,Rc and Rd). Above the two optimal degradation conditions of secondary ginsenoside can produce single configuration of the target product and overcome the shortcomings of the preparation method of the S and R configuration mixture in the reported literature. It has advantage of higher conversion rate and provid the valuable reference to the preparation of protopanaxadiol–type minor ginsenosides.So far, previous studies reported the methods of preparing protopanaxadiol(PPD) are always to degrade the total ginsenosides, protopanaxadiol-type ginsenosides or the monomeric compounds of them. In order to explore the possibility of the process of direct degradation for protopanaxadiol–type secondary ginsenosides by protopanaxadiol–type ginsenosides in the process of extraction, this paper for the first time attempted to the method of the direct extraction of Panax ginseng stalks and leaves with acid solution. The formation of four kinds of secondary ginsenosid--20(S/R)- ginsenoside Rg3 and Rh2 was studied in the extraction process.The optimal condition for the extraction of protopanaxadiol type secondary ginsenoside from Panax ginseng stalks and leaves by aqueous solution of acetic acid was investigated through single factor test and orthogonal design. Experimental results showed the best extraction as follow:The concentration of HAc(V/V,m L/m L) is 50%;the soild-liquid(m/V,g/m L)is 3:50, 80℃; the degradation time is 1h.Using the best degradation condition of the preparation,the total yield of four kinds of secondary ginsenoside is 33.4%, according to protopanaxadiol-type saponin from Panax ginseng stalks and leaves(Rb1,Rb2,Rb3,Rc and Rd). The yield of 20(S)-ginsenoside Rg3 is 12.3%; The yield of 20(R)-ginsenoside Rg3 is 14.8%; The yield of 20(S)-ginsenoside Rh2 is 3%; The yield of 20(R)-ginsenoside Rh2 is 3.3%. This study has proved that the extraction and degradation can be completed simultaneously and it provides a new idea for the direct access to the protopanaxadiol–type ginsenosides by Panax ginseng stalks and leaves.In addition, in order to ensure the accuracy of the results of the above research, the HPLC determination method of 20(S)-ginsenoside Rg3 and 20(R)- ginsenoside Rg3, 20(S)-ginsenoside Rh2, 20(R)- ginsenoside Rh2 were established and the method was fully validated. The experimental result showed that the linear rang of four kinds of secondary ginsenoside is 0.46 to 0.52μg /m L; the average recoveries are 8.54~102.09%(n=6)and its RSD value is less than 2% under the final selection of chromatographic conditions. The result shows that the method is high accurate, good precise and convenient.In conclusion, this paper first discussed the preparation of ginsenoside 20(R)- ginsenoside Rh2 and 20(S)-ginsenoside Rg3 with the total saponins from Panax notoginseng stalks and leaves as raw material, examined the influence factors and established the optimal conditions. Moreover, this paper explored and confirmed the possibility of the process of direct degradation for secondary saponins by protopanaxadiol-type secondary ginsenosides with Panax ginseng stalks and leaves as raw material. It is a new scientific basis for preparation and apply of secondary ginsenoside.