Study On The Anticancer Effects And Mechanisms Of Ginsenoside Rh2 Octyl Ester In Vitro And In Vivo

Panax ginseng C.A. Meyer (Araliaceae) has been used as a functional food and a nutritional supplement for thousands of years in China. Ginsenosides have been considered as the major bioactive ingredients in ginseng. It has been reported that the health benefits of ginsenosides have a wide spectrum, including antiaging, antitumor, anti-inflammation, and antioxidation activities. However, Rh2 has limited application in the food industry and medical supplies due to its low oral bioavailability. In consideration of this, we speculate that the novel ester derivative of Rh2, the octyl ester of Rh2 (Rh2-O), may enhance the permeability of the blood vessel walls for better absorption of Rh2 into tissues. However, the antitumor effects, the underlying mechanism of absorption and antitumor in vitro and in vivo for Rh2-O remains unclear.In the present study, Rh2-O was synthesized by Rh2 and octanoyl chloride. Subsequently, the transepithelial transport and absorption mechanisms of Rh2 and its derivative were studied in the Caco-2 system; the cytotoxicity and cellular uptake of Rh2-O to HepG2 cells and the molecular mechanisms responsible for the anticancer activity of Rh2-O using HepG2 as a representative cell line model were systematically investigated compared with Rh2; the in vivo antitumor activity, immunomodulatory properties, and the possible mechanisms of orally administered Rh2 and Rh2-O were investigated using an animal model (H22 tumor-bearing mice). The main research results obtained in this dissertation are concluded as follows:1. The obtained product from the esterification reaction was identified by MS as an octyl ester derivative of Rh2 (Rh2-O) with m/z 771.6 [M+Na]+. The fatty acid ester substituent was connected to the hydroxyl group at C-12 of Rh2.2. The transport of the ginsenoside Rh2 across the intestinal epithelium was evidenced as very poor, whereas its esterified derivative Rh2-O was verified to possess a better absorption in vitro. The transport rate for apical-to-basolateral (AP-BL) flux of Rh2 (0.21 ×10-6 cm/s) was enhanced by the synthesis of its esterified derivative Rh2-O (1.93×10-6 cm/s) over the concentrations of 10-50 ?M. Verapamil had a significant effect on the Papp values of Rh2 and a negligible effect on that of Rh2-O, the transport mechanisms for both Rh2 and Rh2-O across Caco-2 monolayers were speculated to be transcellular passive diffusion, with an active efflux pump (P-gp) involved for Rh2 and without an active efflux involved for Rh2-O.3. Rh2-O exhibited dose- and time-dependent inhibitory effects against the proliferation of HepG2 cells. The IC50 value of Rh2-O for inhibition of HepG2 cell proliferation was 20.15 ?M, which was roughly half the value of Rh2. Both compounds showed no significant cytotoxicity on HL-02 cells (88.37%) at 25 ?M on HL-02 cells.4. Rh2-O-induced G1 phase arrest was accompanied by the down-regulation of cyclin D3 and cyclin E and cyclin-dependent kinases (CDK) 4 and 6 and the up-regulation of p21WAF1/CIP1 and p27KIP1. In addition, the disruption of Akt and p38 MAPK cascades played a pivotal role in Rh2-O-induced G1 phase arrest.5. Both the extrinsic death receptor pathway and the mitochondrial-mediated intrinsic apoptotic pathway were involved in Rh2- and Rh2-O-induced HepG2 cells apoptosis. The release of lysosomal proteases was upstream of mitochondrial membrane permeabilization.6. Both Rh2 and Rh2-O could significantly inhibit the growth of tumor transplanted in mice compared with the negative control group. No significant toxicity and side effects were observed in the mice treated with Rh2 and Rh2-O. The antitumor effect of Rh2-O(10 mg/kg) was better than that of Rh2 (10 mg/kg) with inhibitory rates of 50.6% and 28.2%, respectively (p<0.05).7. Both Rh2 and Rh2-O induced tumor cell apoptosis by regulating the expression of Bcl-2 family proteins and the activation of caspase family proteins. Additionally, Rh2-O was more efficient than Rh2 in improving immunity system by increasing the serum IL-2 levels, TNF-? production, T lymphocyte counts, CD4+/CD8+ ratio, and NK cell levels. Furthermore, immunehistochemical analysis demonstrated that Rh2 and Rh2-O suppressed tumor angiogenesis by inhibiting VEGF production.8. The antitumor activities of Rh2 and Rh2-O might involve similar mechanisms.The better antitumor activity of Rh2-O than of Rh2 was probably due to its higher intestinal absorption.