The Modification Of Rare Ginsenoside By Fatty Acid And Its Biological Activity

Strong anti-tumor activitives of rare saponins in ginseng were showed in recent pharmacological studies. Reguler ginseng saponins, such as ginsenoside Rg1, Re, Rb1, Rb2, Rc, Rd were gradually de-glycosylated and then converted into rare saponins by the intestinal bacterial metabolism in human body. With the metabolism, ginsenosides 20(S)-panaxatriol group saponins in ginseng mainly convert into ginsenoside Rh1, F1 and 20 (S)-PPt. 20(S)-protopanaxadiol group ginsenosides mainly produce ginsenoside M1, Mc, F2, and 20 (S)-PPd. With the low polar property, rare saponins are easily absorbed by human body and distrubuted in liver through the completion of the blood circulation. Pharmacology studies found that after the fatty acid esterification of rare saponins in liver, finally, rare ginseng saponin fatty acid ester were gotten which showed less toxicity and stronger anti-tumor activitives with longer metabolism time.Ginsenoside Rh2 was first detected in red ginseng by scholars from Japan in 1983. Since then, many pharmacokinetics studies had been done on its pharmacology properties by domestic and abroad scholars. The results illustrated that the oral bioavailability of ginsenoside Rh2 (about 16%) is significantly higher than ginsenoside Rg3 (3%). Li Ke from Shenyang Pharmaceutical University found that Rg3 was mainly distributed in mice liver where it was metabolic transformated into Rh2 and aglycone after administration. Pharmacological studies show that ginsenoside Rh2 have significant inhibition effects on cancer cell proliferation and can induced apoptosis and even kind of stronger than that of ginsenoside Rg3. Based on this, fatty acid ester structure modification of ginsenoside Rh2 in vivo and in vitro were carried out in this study.Firstly, in order to save separation cost, ginsenosides 20(S)-panaxatriol group and 20(S)-protopanaxadiol group saponins were separated by combined using NKA-12 and AB-8 resin.NKA-12 sresin were use in separating the crude saponins in ginseng stem extract and AB-8 macroporous resin were used to separate the two ginseng saponin groups which were the base materials for the subsequent modifion studies.This paper evaluated the transformation rules of ginsenosides in three aspects which include alkaline hydrolysis, enzymatic conversion and thermal processing for obtaining rare ginsenosides. a) Conversion of alkaline solution:It was found that rare ginsenoside Rh1,Rh2, Rh3, S-PPd, S-PPt, F2, Rg5 and so on can be prepared using alkaline hydrolysis. The preparation methods of ginsenoside 20 (S)-Rh1, ginsenoside 20 (S)-Rh1, ginsenoside 20 (S)-PPd, ginsenoside 20 (S)-PPt were optimized in this paper and the four compounds were prepared with conversion rates above 30%. b) Enzymatic conversion:the ginsenosides transformation capacities of snail enzyme were examined. The results showed that although the enzymatic conversion products were complex, ginsenoside CK, Mc, Mx, CY, CO, F1, Rh1 and other rare ginsenosides can be gotten when other methods mentioned above can not got those ginsenosides.c) Thermal processing method (black ginseng's processing):By HPLC-MS method, black ginseng extract were found rich in rare saponins such as ginsenoside Rg3, Rgs, Rh4, Rk1, F4, Rk3 which were the products of thermal process in this study. Besides, the traditional process method for making black ginseng through nine times steaming and nine times driness was optimizated. The results showed that 4-6 times procedures can get the highest rare saponins in making black ginseng which greatly save the time and cost in black ginseng's producting.In this paper, two compounds were synthesized with ginsenoside Rh2 as the substrate, octanoyl chloride as acylating reagent and triethanolamine as the catalyst reagent. The two compounds were identified as C-Rh2 (octanoyl link in the nucleus C-12-bit) and D-Rh2 (octanoyl link in the nucleus C-12-bit and Glycosides C-6 bits) by modern spectroscopy (ESI-MS,1HNMR, 13CNMR, HMBC, HMQC) methods, respectively. In the modification studies, it is found that due to the significantly reduction of C-12 steric hindrance when there was no glucose link in the C-20, the acylation occured preferentially in the C-12 bit of the nucleus. In this study, a synthetic method with relatively high ginsenosides C-Rh2 (46.3%) and D-Rh2 (67.3%) conversion rates determinated by HPLC was established.The anti-tumor activities of modified ginsenoside products C-Rh2 and D-Rh2 in vivo and in vitro were evaluated in present study. The inhibition actvities of compounds C-Rh2, D-Rh2 and Rh2 on HepaG2 tumor cells in vitro were determined by MTT method. The IC50 of Rh2 was 7.40μM and C-Rh2 was 18.10μM and D-Rh2 was above 40μM, respectivly, which were not as good as ginsenoside Rh2. On the other hand, in the anti-tumor studies, the three compounds showed strong anti-tumor activities. The tumor inhibition rate in H22 tumor body bearing mice of ginsenoside Rh2 was 71.1% which was close to the positive anti-tumor drug cyclophosphamide (CTX) (76.6%). Both C-Rh2 and D-Rh2 have inhibitory rate were above 40%. Meanwhile, the inhibitory rate of high doseage C-Rh2 group was 67.1% which repreasents that the anti-tumor activity of C-Rh2 was smilar to the positive drug. Compare with the anti-tumor drug CTX which has strong effect on the immune thymus index, C-Rh2 was found have smaller effect on the immune indices.The effect of ginsenoside Rh2, C-Rh2 and D-Rh2 on diabetic mice were eaxmed using diabetic model mice induced by alloxan. After 14 days of treatment, compared with model group, the blood glucose value of C-Rh2 ginsenoside group with low dose decreased by 42.5% (P<0.05), D-Rh2 ginsenoside group with high-dose decreased by 38.4%(P<0.05), D-Rh2 ginsenoside group with low-dose decreased by 31% and C-Rh2 ginsenoside group with high dose decreased by 24.8%. However, the blood glucose of ginsenoside Rh2 group neither with high nor low doseage was decreased, and there were three mice died during the treatment in the two groups. Compared with the model group, glucose tolerance of C-Rh2 group and D-Rh2 group were significantly improved. The AUC of C-Rh2 high dose group reduced by 28.7% (P<0.05), C-Rh2 low-dose group reduced by 34.7%(P<0.01), D-Rh2 high dose group reduced by 19.2% and D-Rh2 low-dose group decreased by 29.6%(P<0.05), while the ginsenoside Rh2 treatment group showed no decreases in AUC. The positive hypoglycemic insulin showed very obvious blood glucose reduce effects in this study, within 30min after administration the blood glucose decreased to 2.36mmol/L, after 60 min the levels of blood glucose were below 2.2 mmol/L, and no blood glucose increase were seen within 120min. In general, the modification products of ginsenoside Rh2 can be the potential drug for diabetes aid.The anti-tumor and adjuvant treatment of diabetes mellitus effects of ginsenoside have become a hot research. Ginsenosides have been well accepted based on the extensive biological activity characteristics. In this study, low-polarity synthetic derivatives of ginsenoside Rh2 showed strong anti-tumor and anti-diabetic effects. This study provides evidence for their high medicinal values and application prospects in tumor treatment and anti-diabetic.