Analysis of ginesenosides

Abstract: New analytical methods were developed in order to evaluate commercial ginseng preparations and to allow studies on urinary excretion of ginsenosides in humans after consumption of ginseng preparations. An optimized alkaline procedure was developed for cleavage of the glycosidic bonds that was clearly superior to previously reported hydrolysis procedures of ginsenosides because of (a) completeness of the reaction and (b) absence of the artifacts found in acid hydrolysis. The reaction was found to be oxygen-dependent, and almost no reaction occurred in a nitrogen atmosphere. When the air in the reaction medium was replaced with oxygen, the cleavage became complete. Antioxidant, glucose or water markedly inhibited the oxidative cleavage. It was shown that the glucose moiety of glucosides was extensively oxidized under the conditions employed and that formate and carbonate were formed in a ratio of 5/1. Since alkaline conditions and elevated temperature were essential for the oxidative cleavage of the glycosidic bonds, the mechanism may be regarded as a base catalyzed or activated autoxidation. Both alkaline and acidic cleavage products of ginsenosides were separated, identified, and carefully characterized by HPLC, GC and GC-MS. It was demonstrated, for the first time, that the commonly used acid hydrolysis produced six pairs of C-20-epimer aglycones from ginsenosides. Furthermore, it was found that the 20-S epimers of genuine and artificial aglycones (as TMSi ethers) can be distinguished from the corresponding 20-R epimers by differences in both mass spectral fragmentation patterns and retention times. The GC and GC-MS methods developed for sensitive analysis of ginsenosides were used to determine the aglycones released from ginsenosides in 50 commercial ginseng preparations, purchased from 11 countries. As expected, Siberian ginseng and Brazilian ginseng did not contain S- ppd and S-ppt ginsenosides (the specific and major biologically active components of ginseng). Neither Manchurian ginseng nor "Up your gas" gave detectable amounts of S-ppd and S-ppt. Among the remaining 44 ginseng preparations, the contents of total ginsenosides varied from 2.1 to 20.9 mg per capsule, per tablet or per 10 ml of liquid. However, the composition of S-ppd, S-ppt and OA groups of ginsenosides was relatively constant and the amounts of S-ppd and S-ppt and OA ginsenosides were always in the following order: S-ppd > S-ppt > OA (mean ratio: 100 / 42 / 8). A red ginseng and three liquid formulations were found to contain 6 artificial aglycones (R-ppd, R-ppt, hydrated-R-ppd, hydrated-S-ppd, hydrated-R-ppt and hydrated-S-ppt). With a detection limit at a level of a few ng per ml of biological fluids, the GC-MS method developed for quantification of S-ppd and S-ppt glycosides allowed demonstration for the first time, that the ginsenosides are taken up from the human digestive tract and are dose dependently excreted into urine albeit to a seemingly low extent.