Biotransformation of ginsenosides Rb1, Rg3 and Rh2 in rat gastrointestinal tracts

Background Ginsenosides such as Rb1, Rg3 and Rh2 are major bioactive components of Panax ginseng. This in vivo study investigates the metabolic pathways of ginsenosides Rb1, Rg3 and Rh2 orally administered to rats. Methods High performance liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MS-MS) techniques, particularly liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS), were used to identify the metabolites. Results Six metabolites of Rb1, six metabolites of Rg3 and three metabolites of Rh2 were detected in the feces samples of the rats. Rh2 was a metabolite of Rb1 and Rg3, whereas Rg3 was a metabolite of Rb1. Some metabolites such as protopanaxadiol and monooxygenated protopanaxadiol are metabolites of all three ginsenosides. Conclusion Oxygenation and deglycosylation are two major metabolic pathways of the ginsenosides in rat gastrointestinal tracts.


Background
Panax ginseng (Renshen) is used in Chinese medicines to treat various conditions such as debility, ageing, stress, diabetes, insomnia and sexual inadequacy [1][2][3]. The major bioactive components of P. ginseng are O-glycosides of the triterpen dammarane saponins known as ginsenosides [4,5] which exhibit properties such as antiinflammation and anti-tumor [6][7][8]. Over 80 ginsenosides have been isolated from P. ginseng [9]. Rb 1 , Rg 3 and Rh 2 are three major ginsenosides with various bioactivities. Rb 1 , which is the most abundant (0.22-0.62%) among all ginsenosides [5], protects against free radical damage, maintains normal cholesterol and blood pressure [10] and inhibits the induction phase of long-term potentiation by high frequency stimulation in the dentate gyrus of the brain [11]. Rb 1 also rescues hippocampal neurons from lethal ischemic damage [12] and delays neuronal death from transient forebrain ischemia in vitro [13]. Rg 3 is used as the major active component in an anti-tumor and anti-cancer drug in China [14]. The cytotoxicity of ginsenoside Rg 3 against tumor cells increases when Rg 3 is metabolized into Rh 2 or protopanaxadiol [15]. The metabolic transformation of Rg 3 into protopanaxadiol also increases the activity against Helicobacter pylori. Recently, in vitro biotransformation of ginsenosides was reported. The metabolites were identified by high-resolution tandem mass spectrometry. Degradation and bioconversion routes of the different ginsenosides at acidic (gastric) conditions and in the presence of intestinal microbiota were elaborated [16].
High performance liquid chromatography (HPLC) is a powerful chemical analysis technology that allows complex mixtures to be transformed into separated components. Mass spectrometry (MS) has progressed extremely rapidly during the last decade; especially in production, separation and ejection of ions, data acquisition and data reduction. Compared to other detectors, the advantages of the mass spectrometer are that in many cases it can provide absolute identification, not only structural information from the molecule under investigation but the molecular weight of the analyte.
Due to the specificity and sensitivity of LC-MS, especially in combination with MS-MS, it is powerful in identification of drug metabolites. Common biotransformation, e.g., oxidative reactions (hydroxylation), conjugation reactions to produces sulphates, glucuronides, glutathiones or other conjugates, hydrolysis of esters and amides, and reduction reactions, can be evaluated from just the knowledge of the molecular mass of the metabolites. Combination of the molecular-mass and possible biotransformation products, predicted by computer-aided molecular modeling approaches, enables the confirmation of metabolic pathways. Further confirmation and/or structure elucidation of metabolites is possible using MS-MS methods [17]. The identification of the metabolites of antihistamine compounds is feasible by using thermospray LC-MS and LC-MS-MS [18,19]. The present study aims to investigate the biotransformation of ginsenosides Rb 1 , Rg 3 and Rh 2 orally administered to rats by using LC-MS and MS-MS.

Chemicals
Ginsenosides Rb 1 , Rg 3 and Rh 2 (purity >99%) were provided by the Chinese Medicine Laboratory, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, China. HPLC-grade methanol was purchased from Acros Organics (USA). A Mili-Q Ultra-pure water system (Millipore, USA) was used to provide water for all the experiments. Other chemicals (analytical grade) were purchased from Sigma (USA).

Administration of ginsenosides
Water soluble Rb 1 , Rg 3 and Rh 2 were administered to three groups (n = 3 in each group) of male Sprague Dawley rats (body weight 200-220 g; age 6-7 weeks) respectively at a dose of 100 mg/kg body weight with 2 ml dosing solution. The protocols of the animal study were fully complied with the University policy on the care and use of animals and with related codes of practice. The animal experiments were conducted with the licenses granted by Hong Kong Hygiene and Health Department. Rat feces samples were collected at such intervals: 0 to 120 hours for Rb 1 (half-life 16.7 hours), 0 to 24 hours for Rg 3 (half-life 18.5 minutes) and 0 to 48 hours for Rh 2 (half-life 16 minutes) [20][21][22].

Feces sample preparation
Each feces sample of each rat was suspended in 150 ml of water and then extracted with n-butanol (100 ml × 3). The extract was dried and the residue was dissolved in 1 ml of methanol. After centrifugation at 12000 rpm for 20 minutes (Eppendorf Centrifuge 5415R, Hamburg, Germany), 2 μl of the supernatant was analyzed with LC-Ms and LC-MS-MS for the identification of the ginsenosides and their metabolites. The blank feces (baseline) were collected from the same Sprague Dawley rat prior to the administration of ginsenosides, prepared and analyzed with the same method as the experimental groups.

LC-ESI-MS analysis
HPLC separation was performed with a LC system coupled with an auto-sampler and a micro mode pump (HP1100, Agilent Technologies, USA). A reversed-phase column (Waters, Xterra MS-C8, 2.1 × 100 mm, 3.5 μm) was used to separate the ginsenosides and their metabolites. The auto-sampler was set at 10°C. Mobile phase consisted of two eluents: water (A) and methanol (B). For both positive and negative ion mode, the ion source gas 1 (GS1), gas 2 (GS2), curtain gas (CUR) and collision gas (CAD) were 20, 15, 25 and 3, respectively. The temperature of GS2 was set at 400°C.

Metabolites of Rb 1 in rat feces
The parent Rb 1 and direct oxygenated metabolites of Rb 1 were not detected in the feces samples. These results suggested that Rb 1 might have largely metabolized in the gastrointestinal tracts in rats. Six metabolites were detected in rat feces samples collected 0-120 hours after Rb 1 was orally administered (Figure 1). The metabolites were detected from the LC-MS analyses and confirmed by the results from the LC-MS-MS experiments in positive ESI mode [18]. A total of four deglycosylated metabolites were identified, namely Rd, Rg 3 , Rh 2 and protopanaxadiol (        The deglycosylated metabolites were also confirmed by the LC-MS analysis of authentic standards of Rd, Rg 3 , Rh 2 and protopanaxadiol. Moreover, the LC-MS-MS analysis indicated that these deglycosylated metabolites were subsequently oxygenated in digestive tracts. Thus, deglycosylation and subsequent oxygenation are the major metabolic pathways of orally administered Rb 1 in rats. Figure 1 illustrates the proposed metabolic pathways of Rb 1 .

Metabolites of Rg 3 in rat feces
Six metabolites were detected in rat feces samples collected 0-24 hours after Rg 3 was orally administered. The same LC-MS and MS-MS method as for Rb 1 was used to detect major deglucosylated and further oxygenated metabolites of Rg 3 . The MS-MS results were similar to those for Rb 1 . Rh 2 and protopanaxadiol as the deglucosylated products were also confirmed by reference standards. Figure 4 summarizes the major metabolites of Rg 3 detected in the rat feces samples and the metabolic pathway in rat gastrointestinal tracts. After the oral adminis- -glc tration, oxygenation and deglycosylation appeared to be the major metabolic pathways of ginsenosides. Metabolites were detected for the parent Rg 3 and its deglucosylated metabolites including the mono-and deoxygenated products of protopanaxadiol.

Metabolites of Rh 2 in rat feces
Three major metabolites were detected in rat feces samples collected 0-48 hours after Rh 2 was orally administered. The LC-MS and MS-MS method in positive ESI mode was used to detect and confirm the metabolites respectively. Oxygenated products, such as mono-oxygenated protopanaxadiol, were also identified. Deglycosylation and oxygenation were the major metabolic pathways of Rh 2 . Figure 5 illustrates the proposed metabolic pathway of Rh 2 in rat gastrointestinal tracts.

Conclusion
Oxygenation and deglycosylation are two major metabolic pathways of the ginsenosides in rat gastrointestinal tracts. Furthermore, Rh 2 is a metabolite of Rb 1 and Rg 3 , whereas Rg 3 is a metabolite of Rb 1 . Some metabolites such as protopanaxadiol and monooxygenated protopanaxadiol are metabolites of all three ginsenosides.