Lung Cancer Chemoprevention of Ginsenosides is Mediated by the Glycosidases Activities of the A/J Mouse Intestinal Microbiome Defined by Pyrosequencing

[Purpose] Red ginseng extract (RGE) has been reported to possess non-organ-specific preventative effects against several types of cancer, including lung cancer. The ginsenosides responsible for the activity of RGE are poorly defined as primary ginsenosides are inactive. Since most of the primary ginsenosides need the action of bacterial glycosidases to become active one, an important question is if functional activity of bacterial glycosidases would be altered following RGE administration. A more intriguing question is if and how RGE treatment can affect the growth of certain bacteria population in the intestinal microbiome, and whether alteration of such bacteria population would impact the chemoprevention efficacy of RGE. Therefore, the central hypothesis of this thesis is that the lung cancer chemoprevention of ginsenosides is mediated by the glycosidases activites of the A/J mouse intestinal microbiome defined by pyrosequencing. To test the central hypothesis, three specific aims are i) to characterize microbiota dependent metabolism of RGE in A/J mouse fecal lysate; ii) to determine the impacts of RGE administration on bacterial glycosidase activity and intestinal microbiome in A/J mouse; iii) to purifiy and identify bacterial glycosidase(s) from A/J mouse feces that catalyze the rate limiting step in the production of ginesnoside Compound K (one of the most active ginsenosides in RGE). [Methods] The kinetics of microbiota mediated biotransformation of ginesnosides was characterized and kinetic parameters (metabolite formation rates) were determined. The anti-proliferative activity of ginsenosides was tested using the mouse lung cancer LM1 cells. Permeabilities of ginesnosides were also evaluated in Caco-2 cell monolayers. The dose dependent enzymatic functions was tested by giving RGE orally at three doses (daily oral gavage at 5, 50, and 500 mg/kg respectively). The impacts of RGE administration on bacterial glycosidase and intestinal microbiome of A/J mice were evaluated by giving RGE daily to A/J mice for 7 days followed by measuring kinetic parameters (metabolite formation rates), diversity metrics (α diversity and β diversity) and relative abundance of bacteria in the gut microbiome. Bacterial glycosidases were enriched from A/J mouse feces by a classic protein chemistry approach. The identity of the enzymes was examined by LC-MS/MS analysis followed by gene synthesis, molecular cloning, and expression of the enzymes. The functional activity of the enzymes against ginsenoside Rd was tested. [Results] I) Compound K exhibited higher anti-proliferative activity (IC50 ~13 µg/mL) and better permeability (1 ×10-6 cm/s) than primary ginsenosides. Primary ginsenoside Rb1 was converted to Rd, F2, and then Compound K by A/J mouse fecal lysate in a stepwise fashion. Formation of F2 from Rd (metabolite formation rate 0.09 ± 0.003, 0.09 ± 0.01 nmol/min/mg at 20, 5 μM substrate concentration respectively) was the slowest step in the biotransformation of Rb1 to Compound K. II) Bacterial glycosidase activity in response to RGE treatment exhibited a dose dependent manner and the optimal dose of RGE was found to be 50 mg/kg. Oral administration of RGE at 50 mg/kg for 7 days significantly enhanced glycosidase activity of A/J mice by a markedly change (p