Sulfated Modification Of Ginsenoside And Immunological Activities Of Ginsenoside And Its Derivatives

Ginsenosides are the principal active components of many kinds of Panax, and their structure is mainly consisting of sugar and aglycone. Sugar-chain moiety is important position in ginsengoside to exert the biological activity from structure-function relationship. For example, one sugar less than Rg3, Rh2 has stronger anti-tumor activity, while one sugar more than Rg3 at C20, Rd does not have the anti-tumor activity. Some researchers find that ginsenosides, after being metabolized by liver, are changed into fatty acid ester with stronger anti-tumor activity. Enzymolysing the sugar chain of ginsenosides to obtain Rh2 and CK with stronger anti-tumor activity has been the investigative focus of chemical modification.Latest studies showed that related biologic activities were significantly enhanced after certain hydroxy groups of polysaccharides were sulfated. So we would modify the structure of gensonsides at hydroxyl. Otherwise, the less are there sulfate groups on the sugar chain of natural holothurian saponin, the stronger is the anti-tumor activity. So sulfated modification of ginsenoside is possible.Based on the program of the sulfated modification of polysaccharides, the method of the sulfated modification of ginsenoside was explored. The effects of the sulfated derivatives of Ginsenoside on the immunological competence were investigated from in vivo and in vitro aspects, which provided data and new idea for further optimizing molecular structure and searching derivatives with stronger biological activity.Sulfated modification of total saponin of panax ginseng (TSPG) was investigated with the program of the sulfated modification of polysaccharides, the chlorosulfonic acid-pyridine assay, and then the derivatives were identified. The results showed that TSPG was modified successfully and the optimum condition was: V(chlorosulfonic acid):V(pyridine)=1:2.5, reaction time 2 h, reaction temperature < 45℃.Based on the TSPG modification program, Rh2 was sulfated using scheme 1. Besides the characteristic absorption band of Rh2, the infrared spectra of derivatives showed that it also included the absorption peak of sulfate ester bond of C-O-S at 810 cm-1 and that of S=O at 1230 cm-1. The ultraviolet spectra showed that the derivatives had the same absorption band as Rh2 at 196 nm. The mass spectra showed that 1 to 5 -SO3Na were displaced into the structure of Rh2. Two new compounds of sulfated ginsenoside Rh2 were isolated by HPLC and opposite direction silica gel column chromatorgrapy. Their purity is 90% and 91.5% by HPLC, respectively. The IR spectra of the derivatives show the characteristic absorptions of sulfate ester bond. MS spectra of the derivatives show that their molecular masses are 724, indicating that one SO3Na group has been displaced in the structure of ginsenoside Rh2. Nuclear magnetic resonance spectra show that these two new compounds are a couple of iso-merides, they are sodium (8R,10R,14R)-17-((S)-2-hydroxy-6-methylhept-5-en-2-yl)- 4,4,8,10,14-pentamethyl-3-(3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yloxy)hexadecahydro-1H-cyclopenta[a]phenanthren-12-yl sulfate ( Hereinafter abbreviated sulfated Rh2-1,SRh2-1) and sodium (3,4,5-trihydroxy-6-((8R,10R, 14R)-12-hydroxy-17-((S)-2-hydroxy-6-methyl-hept-5-en-2-yl)-4,4,8,10,14-pentameth ylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)tetrahydro-2H-pyran-2-yl) methyl sulfate (Hereinafter abbreviated sulfated Rh2-2,SRh2-2) respectively. By referring to the literature, these two derivatives of Rh2 are new chemical components, which provide material foundation for investigating drugs with stronger biological activity.The effect of TSPG, Rh2 and the derivatives on the proliferation and transformation of lymphocytes was determined by MTT assay, flow cytometry, and morphologic observation. The results showed that TSPG and Rh2 did not have marked effect on the activity of lymphocytes, and inhibited the proliferation of lymphocytes at higher concentration, while the derivatives obviously enhanced the activity of lymphocytes. SRh2-1 had the promoting effect and SRh2-2 had the inhibitive effect under certain condition. The derivatives inhibited the secretion of IL-2 with ELISA assay obviously weaker than TSPG. TSPG promoted or inhibited the secretion of IFN-γdepending on the dose, while the derivatives promoted the secretion of IFN-γ.The effect of ginsenosides and derivatives on the killing activity of NK cell was determined by lactate dehydrogenase releasing assay. The results showed that the derivatives promoted the killing activity of NK cell stronger than TSPG. Rh2 promoted the killing activity of NK cell depending on the dose, while the derivatives had no effect on this activity.To explore the regulating effect of TSPG and the derivatives on the immunological competence of chicken infected by MDV, MD model was established and the changes of leucocytes, proliferation of lymphocytes, the killing activity of NK cell and IFN-γmRNA of peripheral blood lymphocytes were determined. The results showed that TSPG and the derivatives do not change the relative increase of lymphocytes in chicken infected by MDV. TSPG could improve the immunosuppression activity, while the derivatives promote the immunosuppression activity. TSPG and the derivatives could enhance the expression of IFN-γmRNA from lymphocytes in chickens infected by MDV.In a word, we firstly reported the sulfated modification of saponin, and successfully obtained two new compounds after Rh2 was modified, which were a couple of iso-merides, one -SO3Na group being displaced in the structure of Rh2. The effects of the sulfated derivatives of Ginsenoside on the immunological competence were investigated by cell culture, flow cytometry, and RT-PCR from in vivo and in vitro aspects, indicating that the sulfated ginsenoside promoted the immunological competence. These data will provide substantial base for further researches.