Transformation of Ginsenosides from Notoginseng by Artificial Gastric

Article pubs.acs.org/JAFC

Transformation of Ginsenosides from Notoginseng by Artificial Gastric Juice Can Increase Cytotoxicity toward Cancer Cells Jing-Rong Wang,†,‡ Lee Fong Yau,† Rui Zhang,‡,§ Yun Xia,‡ Jing Ma,‡ Hing Man Ho,‡ Ping Hu,§ Ming Hu,# Liang Liu,*,†,‡ and Zhi-Hong Jiang*,†,‡ †

State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Macau 00853, China ‡ School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China § School of Chemistry & Molecular Engineering, East-China University of Science & Technology, Shanghai, China # Department of Pharmacological and Pharmaceutical Sciences, University of Houston College of Pharmacy, Houston, Texas, United States S Supporting Information *

ABSTRACT: Multicomponent metabolic profile of notoginseng saponins in artificial gastric juice was qualitatively and quantitatively investigated, showing that ginsenosides were transformed via multiple pathways including deglycosylation, dehydration, hydration, and oxygenation. A total of 83 metabolites was identified by using UPLC-Q-TOF-MS, among which 16 new dammarane glycosides were further characterized by comparing with synthesized authentic compounds. Transformation time-course of notoginseng saponins in artificial gastric juice was quantitatively measured for the first time, showing rapid degradation of primary ginsenosides and concomitant formation of deglycosylation, hydration, and dehydration products. It was further demonstrated that the resultant metabolites exhibited enhanced cytotoxicity toward cancer cells. The extensive metabolism of ginsenosides within a transit time span in stomach, together with the formation of metabolites with diversified chemical structures possessing enhanced biological activities, indicated an important role of transformation in gastric juice in the systematic effects of ginsenosides. KEYWORDS: notoginseng, ginsenoside, gastric juice, transformation, LC-MS

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INTRODUCTION Roots of Panax notoginseng (Burk) F. H. Chen (Araliaceae), commonly referred to notoginseng and “San-Qi”, is a wellknown medicinal herb in traditional Chinese medicine. It has been historically used as a tonic and hemostatic agent for the treatment of cardiovascular disease, inflammation, different body pains, trauma, and internal or external bleeding due to injury. Substantial studies have suggested hemostatic, antioxidant, hypolipidaemic, hepatoprotective, renoprotective, and estrogen-like activities of notoginseng.1 Recently, chemopreventive and antitumor activities of this herb have received increasing attention.2−4 In China, there are more than 300 herbal preparations and traditional Chinese proprietary medicines containing notoginseng which are being manufactured by over 1000 pharmaceutical companies, including a number of well-known products such as “Xushuangtong Injection”, “Yunnan Baiyao”, “Pien Tze Huang”, etc. Apart from its medicinal application, notoginseng is also a well-known functional food because of its health benefits. It is widely consumed in China and other Asian countries in the form of tea, powder, capsule, and common botanical ingredients in dietary supplements and functional foods. A variety of notoginseng products are available as overthe-counter dietary supplements in the health food market in the United States.5,6 The time-honored application in traditional Chinese medicine, broad usage in pharmaceutical manufacturing, and increasing consumption as functional © 2014 American Chemical Society

food, make notoginseng the most largely consumed Chinese herb in China. The estimated demand for this herb was about 7000 tons every year, even much larger than the demand for Asian ginseng (Radix ginseng). As the principal constituents of notoginseng, more than 60 dammarane-type saponins named ginsenosides, notoginsenosides, and gypenosides, have been hitherto isolated and identified.7,8 These dammarane-type saponins are believed to be responsible for most of the observed pharmacological effects of notoginseng.9 Ginsenosides, such as Rg1, Re, Rb1, and Rd, as well as notoginsenosides such as notoginsenoside R1, are the most abundant dammarane-type saponins in notoginseng. Total content of these triterpene saponins is about 100 mg/g in notoginseng, much higher than that in American ginseng (about 50 mg/g) and Asian ginseng (about 20 mg/g).10,11 It is well-known that ginsenosides were poorly absorbed after oral administration, therefore metabolism of ginsenosides have gained great attention as a key area for explaining their pharmacological actions and their contribution to the clinical efficacy of ginseng products. Numerous investigations have been undertaken to elucidate the fate of ginsenosides through the gastrointestinal tract, showing that intact ginsenosides are Received: Revised: Accepted: Published: 2558

December 5, 2013 February 20, 2014 February 20, 2014 February 20, 2014 dx.doi.org/10.1021/jf405482s | J. Agric. Food Chem. 2014, 62, 2558−2573

Journal of Agricultural and Food Chemistry

Article

largely degraded by either acids,12−14 enzymes, or human intestinal bacteria before reaching the circulation system,15−22 resulting in very low bioavailability of intact ginsenosides (Rb1, 0.1−4.4%; Rb2, 3.7%; Rg1, 1.9−18.4%).23,24 The degraded ginsenosides have been further proven to traverse membrane more efficiently than the intact ginsenosides.25,26 Our previous study has shown increased anticancer activity of ginseng extract and total ginsenosides after acidic hydrolysis.27 All these lead to a transition of study from intact ginsenosides to secondary ginsenosides. Metabolism of ginsenosides in the large intestine, where ginsenosides are deglycosylated by colonic bacteria, was found to mediate the in vivo activity of ginsenosides and thereby was suggested to be a key step for activation of primary ginsenosides which themselves were regarded as a “prodrug”.28,29 For example, one of the major intestinal bacterial metabolite compound K (C−K) also called M1 in some papers), which is derived from PPD-type ginsenosides, has been demonstrated to exhibit in vivo and in vitro anticancer activities.28−30 A metabolic pathway via stepwise cleavage of sugar moieties of ginsenosides with the preference of terminal sugar > inner sugar in intestinal bacteria has been well established, and more than 10 kinds of deglycosylated metabolites have been identified.31,32 Many kinds of bacteria were suggested to integratively metabolize ginsenosides.31 However, among the deglycosylated ginsenosides formed in intestine bacteria, only C−K, Rh1, F1, Rg3, and Rh2 were identified in plasma and urine after oral administration of ginseng,15,20,33,34 while many other gastrointestinal deglycosylated products were only found in feces.35 However, as a colonic deglycosylated product produced by gut microflora, C− K appeared in plasma very late (usually 7−10 h after administration) and its plasma level varied greatly in different studies.20,36 Our previous investigation revealed that P-gP mediated efflux was responsible for the poor absorption of C−K.37 Because of the delayed and low absorption, the plasma level of C−K is very likely to be out of the range where a significant physiological effect can be expected. Unlike the metabolism in gut microflora, transformation of ginsenosides in gastric juice occurred early in stomach. The resultant metabolites have great potential to be absorbed within the long transit time in intestine (2−8 h), which is usually a time span during which pharmacological activities were observed.38 However, although previous study demonstrated the absorption of gastric juice derived metabolites, e.g., Rh1, into the circulation of human,33 little information on the extent and time course of this procedure was available. This greatly hindered understanding of the role of gastric juice-catalyzed biotransformation in the holistic metabolism of ginsenosides after oral uptake. Moreover, recent studies have shown that molecular diversity of ginsenosides can be generated under mild acidic condition (AcOH/EtOH 1:1),39 suggesting that ginsenosides can be metabolized through multiple pathways in addition to the well-known deglycosylation pathway. But there is still no systematic investigation on the identification of metabolites of ginsenosides formed in gastric juice. Because transformation of ginsenosides in gastric juice is the first barrier after being taken orally, and the resulting metabolites then undergo absorption and further metabolism in the gut or liver, it is of paramount importance to investigate the metabolism profile of ginsenosides in gastric juice qualitatively and quantitatively. With this aim, we carried out an in-depth analysis of the metabolites of ginsenosides formed in artificial

gastric juice by using ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and chemical synthesis of speculated metabolites.

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MATERIALS AND METHODS

Chemicals and Materials. Ginsenosides R1, Rb1, Rg1, and Re (each purity >98%) were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China); 20(S)-Rg3, 20(R)-Rg3, 20(S)-Rh2, and 20(R)-Rh2 (Each purity >98%) were purchased from Scholar Bio-Tech (Chendu, China). 20(S)-Rg2, 20(R)-Rg2 (purity >98%), Rk1/Rg5, and Rg6/F4 were isolated in our lab and identified by HR-MS and NMR spectroscopies, and the ratio of Rk1:Rg5 and Rg6/F4 was determined to be 2.1:1.0 and 1.9:1.0, respectively, based on 1H NMR. Total ginsenosides of notoginseng (NS-TG), subtotal ginsenosides of protopanaxadiol-type (NS-PPD), and subtotal ginsenosides of protopanaxatriol-type (NS-PPT) were prepared from MeOH extract of notoginseng in our lab by using column chromatography. All chemicals were of analytical grade purity. Preparation of Hydrolysis Sample of NS-TG, NS-PPD and NS-PPT. Each of NS-TG, NS-PPD, and NS-PPT (30 mg) were accurately weighed into a 50 mL centrifuge tube and 20 mL of 0.1N HCl were added. The solutions were then incubated under 37 ± 0.5 °C with a shaking speed of 100 rpm. At the time points of 0, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 h, a 500 μL aliquot of each sample was taken out and neutralized with 10N NaOH, and then diluted 20-fold with 50% MeOH for quantitative analysis. UPLC-Q-TOF-MS Analysis. UPLC was performed using a Waters ACQUITY UPLC system (Waters Corp., MA, USA) which was equipped with a binary solvent delivery system and a sample manager coupled to Bruker MicroTOF mass spectrometer with an ESI source. All the operations, acquisition, and analysis of data were operated by Hystar software (Bruker). The chromatography was performed on an Acquity BEH C18 column (2.1 mm × 100 mm, 1.7 μm). The mobile phase consisted of 0.1% formic acid in Milli-Q water (A) and acetonitrile containing 0.1% formic acid (B). Two optimized UPLC elution conditions as follows were employed: method I, linear gradient from 15% to 25% B at 0−8 min, 25% to 47.5% B at 8−22 min, 47.5 to 85% B at 22−27 min, and finally 85 to 100% B at 27−29 min; method II, linear gradient from 20 to 40% B within 20 min, then from 40 to 100% B at 20−29 min, and maintained at 100% B for 3 min at a flow rate of 0.35 mL/ min. The injection volume was 1 μL for quantitative analysis and 10 μL for metabolites identification. The ESI-MS data was acquired in negative mode and the conditions of MS analysis were as follows: end plate offset, −500 V; capillary voltage, 4500 V; nebulizing gas (N2) pressure, 1.5 bar; drying gas (N2) flow rate, 8.0L/min; drying gas temperature, 200 °C; mass range, m/z 300−1500. For accurate mass measurement, the TOF mass spectrometer was calibrated routinely using sodium formate solution (0.05 mM NaOH solution with 0.05% formic acid in 90:10 proportion of 2-propanol/distilled water) infused at a flow rate of 2.5 mL/h. For quantitative analysis, the sample solutions were analyzed by enhanced MS scan. Ginsenosides and its metabolites were quantitatively analyzed by using extracted ion chromatogram (EIC) of the [M − H]− or [M + HCOO]− ions. The selected ion for EIC of each ginsenoside was as below: Rb1, m/z 1107.5 ([M − H]−); Rc, m/z 1077.5 ([M − H]−); Rb2, m/z 1077.5 ([M − H]−); Rd, m/z 991.5 ([M + HCOO]−); R1, m/z 977.5 ([M + HCOO]−); Rg1, m/z 845.5 ([M + HCOO]−); Re, m/z 991.5 ([M + HCOO]−); 20(S)- and 20(R)-Rg3, m/z 783.5 ([M − H]−); 20(S)- and 20 (R)-Rh2, m/z 667.4 ([M + HCOO]−); Rk1 and Rg5, m/z 765.5 ([M − H]−); 20(S)- and 20(R)-Rg2, m/z 829.5 ([M + HCOO]−); 20(S)- and 20(R)-Rh2, m/z 683.5 ([M + HCOO]−); Rg6 and F4, m/z 811.4 ([M + HCOO]−). Typical calibration curve for quantitation of each ginsenoside in 50% MeOH were shown in Table 1. Chemical Synthesis of Oxygenated Metabolites. Photosensitized oxygenation products of 20(S)-Rg3, 20(R)-Rg3, 20(S)-Rg2, and 20(R)-Rg2 were synthesized following our protocol described 2559

dx.doi.org/10.1021/jf405482s | J. Agric. Food Chem. 2014, 62, 2558−2573


Article

Compounds 16 and 17 [20(S)-Rg2-mo]: White powder. 1H NMR (pyridine-d5, 400 MHz) δ: 0.93, 0.95, 1.18, 1.34, 1.41, 1.52, 1.53, 1.89, 2.13 (3H each, all s), 1.80 (3H, d, J = 6.2 Hz, Me-6″), 3.49 (1H, dd, J = 11.3, 4.8 Hz, H-3), 4.42 (1H, dd, J = 11.6, 6.7 Hz, H-24), 4.92 (1H, s, Ha-26), 5.27, (1H, s, Hb-26), 5.28 (1H, d, J = 6.8 Hz, H-1′), 6.52 (1H, s, H-1″). 13C NMR data are given in Table 2. Compounds 21 and 24 [20(R)-Rg2-mo]: White powder. 1H NMR (pyridine-d5, 400 MHz) δ: 0.94, 0.99, 1.24, 1.37, 1.41, 2.15 (3H each, all s), 1.53 (6H, s), 1.82 (3H, d, J = 5.4 Hz, CH3-6″), 3.49 (1H, dd, J = 11.7, 6.3 Hz, H-3), 4.41 (1H, dd, J = 12.0, 7.0 Hz, H-24), 4.97 (1H, s, Ha-26), 5.30 (1H, d, J = 6.7 Hz, H-1′), 5.31 (1H, s, Hb-26), 6.51 (1H, s, H-1″). 13C NMR data are given in Table 2. Compounds 35 and 36 [20(R)-Rg2 epoxides]: White powder. 1H NMR (pyridine-d5, 400 MHz) δ:0.80, 1.00, 1.20, 1.30, 1.32, 1.40, 1.42, 2.20 (3H each, all s), 1.80 (3H, d, J = 5.4 Hz, CH3-6″), 4.02 (1H, dd, J = 9.0, 4.0 Hz), 5.10 (1H, d, J = 6.7 Hz, H-1′), 6.50 (1H, s, H-1″). 13C NMR data are given in Table 2. Compounds 44 and 52 [20(S)-Rg3-mo]: White powder. 1H NMR (pyridine-d5, 300 MHz) δ: 0.80, 0.95, 0.98, 1.10, 1.29, 1.46 (3H each, all s), 1.89, 1.93 [1.5 H each, s, H-27 of 24(S) and 24(R)], 3.30 (1H, dd, J = 11.4, 3.9 Hz, H-3), 3.92 (1H, m, H-12), 4.44 (1H, dd, J = 12.0, 4.2 Hz, H-24), 4.91 (1H, d, J = 7.5 Hz, H-1′), 4.93 (1H, s, H-26), 5.25 (1H, s, H-26), 5.37 (1H, d, J = 7.5 Hz, H-1″). 13C NMR data are given in Table 2. Compound 45 [20(S)-Rg3 -mo]: White powder. 1 H NMR (pyridine-d5, 300 MHz) δ: 0.82, 0.95, 1.02, 1.11, 1.29, 1.42 (3H each, all s), 1.56 (6H, s, CH3-26, 27), 3.26 (1H, dd, J = 11.7, 4.5 Hz, H-3), 3.93 (1H, m, H-12), 4.92 (1H, d, J = 7.2 Hz, H-1′), 5.39 (1H, d, J = 7.5 Hz, H-1″), 6.03 (1H, d, J = 16.4 Hz, H-24), 6.50 (1H, ddd, J = 15.0, 7.2, 5.4 Hz, H-23). 13C NMR data are given in Table 2. Compounds 46 and 49 [20(R)-Rg3-mo]: White powder. 1H NMR (pyridine-d5, 300 MHz) δ: 0.83, 0.97, 1.02, 1.14, 1.32, 1.44 (3H each, all s, CH3-30, 19, 18, 29, 26, 28), 1.95, 1.97 [1.5 H each, s, CH3-27 of 24(S) and 24(R)], 3.30 (1H, dd, J = 11.4, 3.9 Hz, H-3), 3.98 (1H, m, H-12), 4.31 (1H, dd, J = 12.6, 4.5 Hz, H-24), 4.97 (1H, d, J = 7.8 Hz, H-1′), 4.98 (1H, s, Hb-26), 5.33 (1H, s, Ha −26), 5.42 (1H, d, J = 7.8 Hz, H-1″). 13C NMR data are given in Table 2. Compound 50 [20(R)-Rg3-mo]: White powder. 1H NMR (pyridine-d5, 300 MHz) δ: 0.85, 0.97, 1.06, 1.13, 1.31, 1.45, 1.56, 1.57 (3H each, all s, CH3-30, 19, 18, 29, 28, 26, 27), 3.30 (1H, dd, J = 11.4, 4.2 Hz, H-3), 3.93 (1H, m, H-12), 4.93 (1H, d, J = 7.2 Hz, H-1′), 5.39 (1H, d, J = 7.5 Hz, H-1″), 6.03 (1H, d, J = 15.6 Hz, H-24), 6.31 (1H, ddd, J = 15.0, 8.4, 5.4 Hz, H-23). 13C NMR data are given in Table 2. Compounds 64 and 66 [20(R)-Rg3 epoxides]: White powder. 1H NMR (pyridine-d5, 400 MHz) δ: 0.79, 1.01, 1.22, 1.29, 1.33, 1.45, 1.46, 2.04 (3H each, s), 3.51 (1H, dd, J = 11.0, 5.0 Hz, H-3), 3.74 (1H, m, H-12), 4.09 (1H, dd, J = 9.7, 3.8 Hz, H-24), 4.94 (1H, d, J = 7.2 Hz, H-1′), 5.40 (1H, d, J = 7.5 Hz, H-1″). 13C NMR data are given in Table 2. Cell Culture and Cytotoxicity Assay of NS-TG, NS-PPD, NSPPT, and Their Metabolites at Different Time Points. Exponentially growing cells were plated in 96-well microplate (Becton, Dickinson and Company) at a density of 1.5 × 104 cells (S180) and 5000 cells (Caco-2 and A549) per well in 100 μL of culture medium and were allowed to adhere for 16 h before treatment (for Caco-2 and A549 cells). Serial concentrations of each compound in DMSO (SigmaAldrich) were then added (100 μL per well), and the cells were incubated for 48 h (S180 and A549) or 72 h (Caco-2) at humidified atmosphere containing 5% CO2 at 37 °C. The final concentration of DMSO in the culture medium was maintained at 0.3% (v/v) to avoid solvent toxicity. Cell viability was determined using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl- tetrazolium bromide (MTT) reduction assay. Briefly, MTT solution (10 μL per well, 5 mg/mL solution) were added to each well and incubated for 4 h at 37 °C, and 100 μL of lysing sodium dodecyl sulfate (SDS) were added and kept overnight at room temperature. The optical densities of the resulting solutions were colorimetrically determined at 570 nm using a microplate reader. Dose−response curves were obtained, and the results were expressed

Table 1. Typical Calibration Curve for Quantitation of Ginsenosides in Incubated Samples of NS-TG, NS-PPT, and NS-PPD ginsenosides/ notoginsenoside R1 Rg1 Re 20(S)-Rg2 20(S)-Rh1 20(R)-Rg2 20(R)-Rh1 Rg6 F4 Rb1 Rd 20(S)-Rg3 20(R)-Rg3 Rk1 Rg5 20(S)-Rh2 20(R)-Rh2

regression equation y y y y y y y y y y y y y y y y y

= = = = = = = = = = = = = = = = =

22754x + 6905.5 37258x + 26583 31852x + 56358 21623x+ 12199 69284x + 44700 23808x + 2114.9 30436x + 20330 2901.1x + 642.87 3192.6x − 462.66 18687x + 9683.6 23118x + 31313 29880x + 14483 22808x + 20843 18966x + 25526 19458x + 20165 48494x + 5778.5 51129x + 3367.3

linearity (mg/L)

r2

0.035−33.4 0.040−44.3 0.042−76.5 0.35−50.3 0.5−24.4 0.35−50.3 1.3−196 1.5−237.6 1.5−237.6 0.591−151 0.05−58.3 0.55−28.1 0.35−52 0.8−121.3 0.8−121.3 0.11−21.3 0.13−20.0

0.9976 0.9924 0.9920 0.9954 0.9954 0.9997 0.9989 0.9997 0.9992 0.9955 0.9908 0.9934 0.9952 0.9935 0.9955 0.9991 0.9993

previously.40 Briefly, a solution of the parent compound (200 mg) and Rose-Bengal (20 mg) in MeOH was stirred and irradiated with 400 W lamp under an oxygen atmosphere at room temperature for 10 h. After filtration, triphenylphosphine (40 mg) was added to the filtrate and the solution was stirred at room temperature for another 4 h. The solution was evaporated to dryness in vacuum, then subjected to combinative column chromatographies over silica gel eluted with CHCl3−MeOH (10:0−8:2), MCI-gel CHP 20P, and Bondpack ODS (eluted with 40− 100% MeOH) to give individual oxygenated products. Epoxidation of 20(R)-Rg3 and 20(R)-Rg2 were performed following the procedure described in the literature with minor modifications.41 Briefly, solution of the selected ginsenosides (100 mg) in acetonitrile (10 mL) was mixed with an aqueous EDTA-Na solution (0.4 μM, 10 mL). The mixed solution was cooled to 0 °C, and then an epoxidation dibetal catalyst solution (ketone, 60 μM in acetonitrile) and the mixture of sodium bicarbonate (155 μM) and oxone (100 μM) were added. The homogeneous solution was stirred overnight and then extracted with n-BuOH. The resultant n-BuOH layer was applied to column chromatographies over SiO2 and ODS to afford 20(R)-Rg3 epoxide (compounds 64 and 66, 21.6 mg) and 20(R)-Rg2 epoxide (compounds 35 and 36, 15.02 mg) . Chemical structures of the synthesized metabolites were characterized based on the elucidation of NMR data. 1H- and 13C NMR data were recorded on a Varian Mercury-Plus 300 MHz NMR spectrometer (300 MHz for 1H and 75 MHz for 13C), Varian 400 MHz FT-NMR spectrometer (400 MHz for 1H and 100 MHz for 13C) and Bruker NMR spectrometer (CyroFIT, 600 MHz for 1H and 150 MHz for 13 C). Coupling constants were given in Hz and chemical shifts are represented in δ (ppm) relative to Me4Si as internal standard. HR-ESIMS was performed on a Q-TOF mass spectrometer (Bruker Daltonics, MA, USA). Compound 11 [20(S)-Rg2-mo] White powder. 1H NMR (pyridined5, 400 MHz) δ: 0.96, 1.01, 1.29, 1.30, 1.41, 1.56, 1.58, 2.16 (3H each, all s), 1.82 (3H, d, J = 6.2 Hz, Me-6″), 3.51 (1H, dd, J = 11.2, 4.6 Hz, H-3), 5.30 (1H, d, J = 6.9 Hz, H-1′), 6.08 (1H, d, J = 15.6 Hz, H-24), 6.37 (1H, ddd, J = 15.6, 9.2, 5.5 Hz, H-23), 6.55 (1H, brs, H-1″). 13C NMR data was given in Table 2. Compound 12 [20(R)-Rg2 -mo]: White powder. 1H NMR (pyridine-d5, 400 MHz) δ: 0.95, 1.01, 1.28, 1.37, 1.39, 1.54, 1.56, 2.10 (3H each, all s), 1.78 (3H, d, J = 6.0 Hz, Me-6″), 3.48 (1H, dd, J = 11.2, 4.8 Hz, H-3), 4.35 (1H, m, H-6), 5.25 (1H, d, J = 6.8 Hz, H1′), 6.05 (1H, d, J = 15.6 Hz, H-24), 6.32 (1H, ddd, J = 15.6, 9.4, 5.6 Hz, H-23), 6.47 (1H, brs, H-1″). 13C NMR data are given in Table 2. 2560



Article

Table 2. 13C-NMR Data of the Chemically Synthesized New Metabolites (in Pyridine-d5) C

11a

12a

16 and 27a

21 and 24a

35 and 36a

44 and 52a

45b

46 and 49a

50b

64 and 66a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″

39.1 27.4 78.3 39.8 60.6 74.0 45.8 41.0 49.6 39.5 32.0 70.8 48.3 51.5 31.0 26.5 53.9 17.4 17.4 73.1 26.5 39.4 122.9 141.8 69.6 30.5 30.5 27.5 17.0 16.7 101.6 79.2 78.2 72.1 78.1 62.9 101.7 72.4 72.2 74.2 69.2 18.5

39.7 26.7 78.9 40.3 61.1 74.5 46.3 41.4 50.0 39.9 32.5 71.3 49.3 51.0 31.6 26.7 52.0 17.9 17.5 74.1 22.9 46.4 123.1 142.7 70.2 31.1 31.1 28.0 17.4 17.5 102.0 79.7 78.7 72.6 78.6 63.4 102.3 72.9 72.7 74.5 69.8 19.1

39.6 28.0 78.8 40.3 61.1 74.4 46.3 41.4 50.1 39.9 31.6 71.3 48.5 52.0 30.9 27.1 55.1 (54.8) 18.5 17.9 73.4 (73.3) 24.3 32.5 31.6 76.6 (76.3) 149.7 110.4 (110.1) 18.8 29.4 17.2 17.4 102.1 79.7 78.7 72.5 78.6 63.3 102.3 72.8 72.7 74.6 69.7 19.0

39.1 27.5 78.3 39.8 60.6 74.0 45.8 40.9 49.5 39.4 31.1 70.6 48.6 51.5 29.6 26.4 50.3 (50.1) 17.9 17.4 72.8 22.7 39.1 32.0 75.9 (75.8) 149.6 110.0 (109.8) 18.1 27.5 17.0 17.4 101.5 79.2 78.2 72.1 78.1 62.9 101.8 72.3 72.2 74.1 69.2 18.5

38.6 27.4 79.0 40.5 61.3 74.6 46.5 41.7 50.1 40.2 32.7 71.5 49.8 51.2 32.7 26.4 52.1 (52.0) 19.4 19.2 86.7(86.6) 21.8(21.2) 39.9 (39.6) 27.9 (27.5) 87.5 (86.3) 70.9 (71.2) 26.6 (26.9) 27.2 (28.2) 31.9 18.1 18.0 102.4 79.9 78.9 72.8 78.8 63.6 102.4 73.1 72.9 75.0 69.9 20.0

39.4 27.7 89.2 40.0 56.6 18.4 35.4 40.3 50.3 37.2 30.2 71.3 48.8 52.0 30.2 27.0 55.1 (54.9) 16.8 16.6 73.4(73.3) 27.2 31.6 30.9 76.6 (76.3) 150.5 110.4 (110.1) 18.7 28.4 16.1 17.3 105.4 83.6 78.2 71.9 78.4 62.8 106.3 77.4 78.6 71.9 78.6 62.1

39.6 26.8 89.2 40.1 56.7 18.9 35.6 40.4 50.6 37.5 32.5 70.2 49.8 51.2 31.8 26.8 52.3 16.3 16 74.2 23 46.6 121.5 142.6 70.2 31.2 31.2 28.6 16.8 17.6 105.4 83.7 78.5 71.8 78.6 63.2 106.3 77.4 78.7 71.2 78.3 63.1

39.4 27.0 89.2 40.0 56.6 18.6 35.4 40.3 50.6 37.2 31.6 71.2 49.5 52.0 30.1 26.9 51.0 16.9 16.7 73.3 23.2 39.6 32.4 76.3 (76.4) 150.5 110.6 (110.4) 18.4 28.4 16.1 17.6 105.4 83.7 78.6 71.9 78.4 63.0 106.3 77.5 78.6 71.9 78.2 63.1

39.3 26.9 89 39.8 56.5 18.6 35.3 40.1 50.5 37 32.2 71.1 49 51.8 31.4 26.8 54.2 16.6 16 73.4 27.7 39.8 123.3 141.8 69.9 30.8 30.8 28.3 17.1 16.7 105.1 83.3 78 71.7 78.3 62.9 106 77.1 78.4 71.7 78.1 62.7

39.5 28.4 89.2 40.0 56.6 18.7 35.5 40.3 50.5 37.2 31.4 71.0 49.9 52.0 51.8 26.2 51.2 (51.1) 16.9 16.0 86.5 (86.4) 21.6(19.3) 39.4(38.4) 27.7 (27.3) 87.4 (86.2) 70.9 (70.5) 26.4 (26.8) 30.3 (27.0) 31.7 16.7 17.4 105.4 83.6 78.4 71.8 78.3 63.1 106.3 77.4 78.6 71.9 78.6 63.0

a

150 MHz for 13C NMR; b75 MHz for previously.7,40,46,48,66

13

C NMR. The signals were assigned by comparing with those of reference compounds reported

as IC50 values in μg/mL. Each experiment was carried out for three times. IC50 results were expressed as means ± standard deviation.

type (NS-PPD), subtotal ginsenosides of protopanaxatriol-type (NS-PPT), and total ginsenosides (NS-TG) incubated for 3 h, the time point when most of the primary ginsenosides were transformed, were analyzed by using UPLC-Q-TOF-MS. In the incubated sample of NS-PPT, four groups of peaks can be easily observed as the major metabolites in the total ion chromatogram (TIC). These major metabolites were easily identified as deglycosylated ginsenosides, corresponding dehydration products, hydration products of primary, and secondary ginsenosides, as well as hydration products of dehydrated ginsenosides (Supporting Information). For NS-PPD (Figure 1B), similarly, the above four groups of metabolites were observed and characterized based on the retention time, quasimolecular ion, and accurate mass. Notably, the metabolites profile of NS-PPD was somewhat different from that of NS-PPT. The relative

■

RESULTS Identification of Metabolites Formed in Artificial Gastric Juice. A primary study of NS-TG incubated in 0.1 N HCl and artificial gastric juice prepared following “Chinese Pharmacopoeia” give quiet similar profile in both metabolism pathway and metabolism time course. Further examination of NS-TG incubated in pepsin alone revealed that pepsin does not have any effect on the metabolism of NS-TG (Supporting Information). Therefore, 0.1N HCl was employed for simulating gastric condition in the current study to avoid steps for protein removal. To survey the metabolites formed in artificial gastric juice, subtotal ginsenosides of protopanaxadiol2561



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Figure 1. Total ion chromatogram (TIC) of NS-PPT (A), NS-PPD (B), and NS-TG (C) before (upper panel) and after incubation (lower panel) in artificial gastric juice for 3 h. 2562



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Figure 2. Structures of the oxygenated ginsenosides identified by comparing with synthesized authentic compounds.

Figure 3. Identification of dioxygenated (A), mono-oxygenated (B), and epoxide metabolites (C) of Rg3 in NS-TG incubated for 3 h in artificial gastric juice by comparing with synthesized authentic compounds. From top to bottom: NS-TG before incubation, NS-TG incubated for 3 h, and synthesized authentic metabolites of 20(S)- and 20(R)-Rg3.

abundance of hydrated metabolites in NS-PPD was much lower than that in NS-PPT, while the intensity of dehydrated metabolites was just the opposite. Takino has reported that PPT-type ginsenosides is easily hydrated in both 0.1 N HCl and rat stomach, whereas PPD-type ginsenosides did not undergo hydration in either medium.13,14 Our result showed that hydration of PPD-type ginsenosides also occurs in artificial gastric acid, but to much less an extent than that of PPT-type ginsenosides. On the basis of the identification of metabolites of NS-PPT and NS-PPD incubated with artificial gastric juice, metabolites derived from the primary ginsenosides R1, Rg1, Re, Rb1, and Rd in the incubated sample of NS-TG through the above metabolic pathways were characterized or assigned by using the similar approach (Figure 1C).

Because oxygenated ginsenosides were also reported to form in rat stomach (in vivo) or under mild acidic condition except for the above major metabolic pathways.42 We further screened oxygenated ginsenosides in NS-TG metabolites by using EIC mode in LC-MS. As a result, mono- and dioxygenated Rg3 and Rg2 were detected to be the major oxygenated metabolites of ginsenosides in artificial gastric juice. To identify the oxygenated metabolites, we synthesized speculated authentic compounds through photosensitized oxygenation and epoxidation. Chemical structures of the synthesized compounds were confirmed based on spectroscopic evidence. 20(S)-Rg2mo-I (11), a metabolite synthesized by using 20(S)-Rg2 as the starting material, was taken as an example to show the structure elucidation of photosensitized ginsenosides. The proton and carbon signals of aglycone part in the 1H NMR and 13C NMR 2563



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Table 3. Characterization of Metabolites of NS-TG in Artificial Gastric Juice by Using UPLC-Q-TOF-MSb

2564



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Table 3. continued

2565



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Table 3. continued

a The peak contained two metabolites which can be separated under optimized condition; Abbreviations: N-R2 = notoginsenoside R2; N-T5 = notoginsenoside T5; Hy = hydration products; Degly = deglycosylation products; dio = dioxygenated derivatives, i.e., 24- or 25-hydroperoxides; mo = mono-oxygenated derivatives, i.e., 24- or 25-hydroxides derived from photosensitization followed by reduction; epo = 20,24-epoxide. b*New compounds which were confirmed by comparison with chemically synthesized authentic compounds.

disubstituted olefin as evidenced by the signals at δH 6.08 (1H, d, J = 15.6 Hz, H-24), 6.37 (1H, ddd, J = 15.6, 9.2, 5.5 Hz, H-

spectrum of 11 were found to be very similar to that of 20(S)Rg2 except for some signals arising from the side chain.43,44 A 2566



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Figure 4. Proposed metabolic pathways of ginsenosides in artificial gastric juice.

23), and δC 122.9 (C-23), 141.8 (C-24). The downfield shift of C-22 (ΔδC = 3.5 ppm), compared to that of 20(S)-Rg2, clearly suggested a translocation of the double bond to C-23 and 24. The oxygenated carbon (δC 69.9) assigned to the remaining carbon (C-25) of the side chain indicated the presence of a hydroxyl group at C-25. These evidence revealed the structure of the side chain with a hydroxyl group at C-25 and a double bond at C-23 and C-24, which was supported by the chemical shift comparing with those of related hydroxylated ginsenosides.7 Thus the structure of 11 was assigned as shown (Figure 2). Structure characterization of epoxides was shown in example of 20(R)-Rg2-Epo (35 and 36). This mixture was synthesized from 20(R)-Rg2 by using epoxidation dibetal catalyst solution which could oxidize 24,25-double bond into 24,25-epoxide. The resulting 24,25-epoxy compounds are quite unstable in the existence of a 20-hydroxy group and immediately changed into 20,24-epoxides.45 The formation of 20,24-epoxides in 35 and 36 was evidenced by the large downfield shift of C-20 (ΔδC = 13.3) and C-23 (ΔδC = 5.3), as well as the displacement of olefinic carbon signals with oxygenated carbon signals [δC 87.5(C-24), δC 70.9(C-25)] in comparison with that of 20(R)-type secondary ginsenosides,14 and was further supported by comparing the chemical shifts to those of 20(S)epoxides.46 35 and 36 were found to be an equivalent mixture of 24(R) and 24(S)-epimers based on the fact that the signals of C-17,20−27 appeared in pairs in the 13C NMR spectrum (Table 2). On the basis of above evidence, the structure of 35 and 36 was assigned as shown (Figure 2).

By comparing with the synthesized metabolites, the dioxygenated metabolites were identified to be hydroperoxides derivatives of Rg2 and Rg3, while the mono-oxygenated metabolites were characterized to be corresponding hydroxides and 20,24-epoxides (Figure 3) of Rg2 and Rg3. Hydroperoxides and corresponding hydroxides of Rg2 and Rg3, as well as epoxides of 20(R)-Rg2 and 20(R)-Rg3, are new dammarane glycosides which were not reported before. To confirm formation pathway of the oxygenated metabolites, ginsenoside Rg3 was incubated in artificial gastric juice, resulting in the production of above oxygenated metabolites (data not show), indicating these oxygenated metabolites can be derived from Rg3 via oxygenation rather than from hydrolysis of hydroperoxides and corresponding hydroxides of primary ginsenosides that have been reported to exist naturally in notoginseng.47,48 In summary, totally 83 metabolites were identified in the incubated sample of NS-TG (Table 3). Characterization of these metabolites suggested deglycosylation, dehydration, hydration, and oxygenation as the metabolic pathways of ginsenosides in artificial gastric juice (Figure 4). Time Course of Transformation of Ginsenosides in Artificial Gastric Juice. The time courses of degradation of primary ginsenosides, as well as the formation of deglycosylation and dehydration metabolites, were plotted by quantitatively determining the content of both parent compounds and metabolites at different time point of incubation (Figure 5). It can be seen that the three primary ginsenosides, N-R1, Rg1, and Re, disappeared rapidly. N-R1 and Re were exhausted at 2 h, while Rg1 was metabolized thoroughly at 3 h (Figure 5A,B). 2567



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Figure 5. Time course of degradation of primary PPT-type ginsenosides (A,B), formation of deglycosylation, dehydration (C,D), and hydration metabolites (E,F) in incubated NS-TG and NS-PPT. Each sample was analyzed in duplicate, and the average value was employed for plotting.

Formation of the dehydration products, e.g., Rg6 and F4, showed almost identical time course with that of corresponding secondary ginsenosides, and the ratio between them kept the same during 8 h incubation, suggesting that dehydration occurred very quickly after deglycosylation and reached equilibration rapidly. PPD-type ginsenosides showed quite similar time course to that of NS-PPT (Figure 6A−D), but

20(R)- and 20(S)-Rh1 were the most dominant metabolites, followed by 20(S)- and 20(R)-Rg2 and their dehydration products Rg6 and F4. Production of the metabolites peaked at 2 h, and decreased stepwise after 2 h (Figure 5C,D). Decrease of the deglycosylated and dehydrated metabolites might be due to subsequent hydration, which was supported by the increased formation of hydration products after 2 h (Figure 5E,F). 2568



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Figure 6. Time course of degradation of primary PPD-type ginsenosides (A,B) and formation of deglycosylation and dehydration metabolites (C,D) in artificial gastric juice. Each sample was analyzed in duplicate, and the average value was employed for plotting.

Table 4. Cytotoxicity of NS-TG, NS-PPT, NS-PPD, and Their Metabolites Formed at Different Time Point of Incubation in Artificial Gastric Juice Towards S180, Caco-2, and A549a content (μg/mg)

ratio of different types of ginsenosides

sample

PG

SG

DG

SG/PG

DG/PG

(SG + DG)/PG

TG_0 h TG_0.25 h TG_0.5 h TG_1 h TG_3 h PPT_0 h PPT_0.25 h PPT_0.5 h PPT_1 h PPT_3 h PPD_0 h PPD_0.25 h PPD_0.5 h PPD_1 h PPD_3 h

905.66 673.74 547.06 287.44 21.00 812.09 428.56 362.54 199.08 4.00 812.13 591.08 483.66 330.93 67.90

25.31 123.00 161.96 234.66 309.71 21.84 182.77 236.50 287.17 274.52 3.86 74.25 117.45 176.51 276.37

3.75 80.31 124.66 204.10 305.91 17.89 44.11 52.87 60.87 55.42 9.51 140.01 217.36 328.62 499.56

0.03 0.18 0.30 0.82 14.75 0.03 0.43 0.65 1.44 68.63 0.00 0.13 0.24 0.53 4.07

0.00 0.12 0.23 0.71 14.57 0.02 0.10 0.15 0.31 13.86 0.01 0.24 0.45 0.99 7.36

0.03 0.30 0.52 1.53 29.32 0.05 0.53 0.80 1.75 82.49 0.02 0.36 0.69 1.53 11.43

IC50 (μg/mL) S180 >2000 449.8 ± 264.2 ± 175.9 ± 133.2 ± >2000 377.2 ± 248.8 ± 229.9 ± 348.6 ± >2000 467.1 ± 242.5 ± 305.4 ± 121.3 ±

13.2 5.2 37.2 16.5 99.5 5.3 17.5 10.5 43.2 5.9 42.3 7.4

Caco-2

A549

>2000 776.6 ± 114.1 302.6 ± 62.0 393.7 ± 92.0 144.5 ± 12.8 >2000 624.9 ± 282.0 337.2 ± 36.0 408.3 ± 49.9 620.5 ± 45.6 >2000 1932.7 ± 38.0 441.7 ± 82.1 665.1 ± 152.6 246.9 ± 0.6

>2000 317.0 ± 49.5 181.8 ± 47.6 183.3 ± 46.7 57.2 ± 12.6 >2000 286.7 ± 44.7 148.2 ± 15.7 153.8 ± 14.2 190.2 ± 5.5 >2000 487.9 ± 10.3 228.8 ± 40.3 115.6 ± 1.3 30.5 ± 0.6

a

Abbreviations: PG = sum of primary ginsenosides (N-R1, Rg1 Re, Rb1, Rd); SG = sum of secondary ginsenosides (Rg2, Rh1, Rg3, Rh2); DH = sum of dehydration products (Rg6, F4, Rk1 and Rg5).

Cytotoxicity of Metabolites of Ginsenosides at Different Time Point to Cancer Cells. Cytotoxicity of ginsenosides

different from PPT-type ginsenosides, concentration of the metabolites did not decrease at the later period of incubation. 2569



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the removal of sugar moiety.54−56 Moreover, it has been reported that deglycosylated ginsenosides, i.e., F2, C−K, Rh2, Rh1, and F1 possessed slightly increased membrane permeability than primary ginsenosides, while aglycone, PPD and PPT further showed 1-fold higher permeability than that of deglycosylated ginsenosides in Caco-2 cells.57 Therefore, deglycosylated metabolites might have great potential to be absorbed into circulation system. This can be exampled by previous report which showed the absorption of gastric juicederived metabolites of ginsenosides (e.g., Rh1 and hydrated Rh1) at early stage (0−4 h) in urine and plasma of human after oral administration of ginseng extract.33 Even though, absorption of the deglycosylated ginsenosides into the circulation system could be extremely complicated because of the efflux and stereoselectivity as well as further metabolism. Actually most of the deglycosylated ginsenosides formed in gastric juice were not reported to exist in plasma or only detected in nanomolar levels.36 A major reason might be the involvement of efflux transport. For example, 20(S)-Rh2, a major deglycosylated PPD-type ginsenosides formed in gastric juice, was demonstrated to be a substrate of P-gP. Its transport was involved by P-gP and thus resulting in poor absorption.58 As C-20 epimers of the deglycosylated ginsenosides formed almost simultaneously, stereoselective absorption as well as stereoselective regulation of P-glycoprotein by Rh2 epimers will result in quite complicated interaction of these two epimers,59,60 making it difficult to predict their absorption. An additional issue potentiate the complexity in absorption of the metabolites is the subsequent metabolism in liver after absorption. Recent studies suggested that C-20 hydroxyl group was a determinant for hepatic disposition of ginsenosides,61 implying that biotransformation of ginsenosides in gastric juice through hydrolysis of C-20 sugar moiety would be a key step for “activating” the subsequent P450-mediated metabolism. Except for the deglycosylated ginsenosides, the corresponding dehydrated and hydrated ginsenosides may also be metabolized in liver to form bulky metabolites. The metabolites resulting from the coupling of gastric juice and hepatic enzyme might be responsible for the unidentified circulating metabolites proposed in previous radioisotope assay, which revealed three times higher serum radioactivity of Rb2 and its derivatives than Rb2 level determined by HPLC.23 It has been proved that intact ginsenosides and deglycosylated ginsenosides exhibited distinct activity on CYP enzymes.62,63 Therefore, systemic exposure of the deglycosylated and related ginsenosides was quite valuable for evaluating the herb−drug interaction caused by the inhibition of ginsenosides on the CYP’s activity. Although substantial studies have demonstrated in vivo anticancer effect of primary ginsenosides,64−66 no in vitro evidence of cytotoxicity of primary ginsenosides was available. Our studies showed that primary ginsenosides themselves seems to be “inactive” toward cancer cells but can gain potent cytotoxicity after biotransformation in artificial gastric juice, suggesting that this step may contribute to the observed in vivo anticancer activity of notoginseng. Moreover, both PPD- and PPT-type ginsenosides exhibited increased cytotoxicity after biotransformation and may cooperatively contribute to the activity of NS-TG metabolites. The cytotoxicity of metabolites enhanced along with the increased incubation time and showed good correlation with the ratio of both deglycosylated and dehydrated products to primary ginsenosides. This evidence indicated a major contribution of deglycosylation and dehydration products to the cytotoxicity. However, other

toward a broad spectrum of cancer cell lines have been demonstrated, hence the most widely used cancer cell lines in our lab, S180, A549, and Caco-2 cells, were selected for the assaying cytotoxicity. Among these three cell lines, A549 and Caco-2 have been reported for evaluating cytotoxicity of ginsenosides.49−51 As shown in Table 4, all the ginsenoside samples before incubation exhibited no cytotoxicity (IC50 > 2000 μg/mL) or very low cytotoxic activities. With the increase of incubation time in artificial gastric juice, cytotoxicity of all three samples enhanced gradually. Cytotoxicity potential of the ginsenosides were found to be closely related to the ratio between metabolites and primary ginsenosides, indicating that secondary and dehydrated ginsenosides are the major active principles responsible for the cytotoxicity toward cancer cells. However, MTT is a colorimetric assay based on tetrazolium dye reduction catalized by NAD(P)H-dependent oxidoreductase. Therefore, viable but metabolically quiet resting cells like thymocytes and splenocytes reduce very little MTT, leading to a false conclusion. More importantly, although MTT assay is widely used for assaying cell viability, it could not distinguish the effects of cell death or cell growth inhibition, which are important component of biological research. Further studies need to be carried out to evaluate the subtle influence of metabolism on the bioactivity of ginsenosides.

■

DISCUSSION Our study revealed multiple metabolic pathways of ginsenosides in gastric juice, including deglycosylation, dehydration, hydration, and oxygenation. The hydration of PPT-type ginsenosides was much more extensive than that of PPD-type ginsenosides, indicating that this pathway is compound typedependent. Oxygenation, which occurs through both epoxidation and photosensitization, was shown to be a minor metabolic pathway of ginsenosides in gastric juice. These results indicated that molecular diversity of ginsenosides can be generated through transformation in gastric juice, thus creating new chemomes which are more biologically associated with the activity of the herb. In a previous study, the hydrolytic reaction of ginsenosides under mild acidic condition (AcOH/EtOH 1:1) led to the formation of deglycosylation product, dehydration product, 20O-alkylated derivatives, 25-OH derivatives, as well as methylation product.39 In our study, deglycosylation, dehydration and hydration product (25-OH derivatives) were found to be the major metabolic pathway of ginsenosides, suggesting that these pathways are common reaction under acidic condition. Time course investigation suggested that the primary ginsenosides were decomposed quickly within 2 h, a period required for exclusion of stomach, implying that ginsenosides undergo extensive metabolism in gastric juice before they get into the intestine in vivo. Deglycosylation products, e.g., Rg3, Rh2, Rg2, and Rh1, were demonstrated to be the major metabolites formed in artificial gastric juice, accounting for 30− 40% of the total amount of metabolites. Previous studies have shown that after oral administration significant amount of C−K can be detected in plasma while the primary ginsenosides were poorly absorbed or undetectable.52,53 Rh2, a deglycosylated ginsenoside derived from artificial gastric acid, was reported to have an improved intestinal absorption as evidenced by much higher bioavailability (∼5% in rat, 15−25% in dog) than its parent ginsenosides Rb1 and Rb2 (with bioavailability of 0.78 and 0.08%, respectively), implying an improved absorption with 2570



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(12) Karikura, M.; Miyase, T.; Tanizawa, H.; Taniyama, T.; Takino, Y. Studies on absorption, distribution, excretion and metabolism of ginseng saponins. VI. The decomposition products of ginsenoside Rb2 in the stomach of rats. Chem. Pharm. Bull. (Tokyo) 1991, 39 (2), 400− 404. (13) Karikura, M.; Miyase, T.; Tanizawa, H.; Taniyama, T.; Takino, Y. Studies on absorption, distribution, excretion and metabolism of ginseng saponins. VII. Comparison of the decomposition modes of ginsenoside-Rb1 and -Rb2 in the digestive tract of rats. Chem. Pharm. Bull. (Tokyo) 1991, 39 (9), 2357−2361. (14) Odani, T.; Tanizawa, H.; Takino, Y. Studies on the absorption, distribution, excretion and metabolism of ginseng saponins. IV. Decomposition of ginsenoside-Rg1 and -Rb1 in the digestive tract of rats. Chem. Pharm. Bull. (Tokyo) 1983, 31 (10), 3691−3697. (15) Wang, Y.; Wang, B. X.; Liu, T. H.; Minami, M.; Nagata, T.; Ikejima, T. Metabolism of ginsenoside Rg1 by intestinal bacteria. II. Immunological activity of ginsenoside Rg1 and Rh1. Acta Pharmacol. Sin. 2000, 21, 792−796. (16) Bae, E.-A.; Han, M. J.; Kim, E.-J.; Kim, D.-H. Transformation of ginseng saponins to ginsenoside Rh2 by acids and human intestinal bacteria and biological activities of their transformants. Arch. Pharm. Res. 2004, 27, 61−67. (17) Bae, E.-A.; Choo, M.-K.; Park, E.-K.; Park, S.-Y.; Shin, H.-Y.; Kim, D.-H. Metabolism of ginsenoside R(c) by human intestinal bacteria and its related antiallergic activity. Biol. Pharm. Bull. 2002, 25, 743−747. (18) Bae, E.-A.; Shin, J.-E.; Kim, D.-H. Metabolism of ginsenoside Re by human intestinal microflora and its estrogenic effect. Biol. Pharm. Bull. 2005, 28, 1903−1908. (19) Hasegawa, H.; Lee, K. S.; Nagaoka, T.; Tezuka, Y.; Uchiyama, M.; Kadota, S.; Saiki, I. Pharmacokinetics of ginsenoside deglycosylated by intestinal bacteria and its transformation to biologically active fatty acid esters. Biol. Pharm. Bull. 2000, 23, 298−304. (20) Akao, T.; Kida, H.; Kanaoka, M.; Hattori, M.; Kobashi, K. Intestinal bacterial hydrolysis is required for the appearance of compound K in rat plasma after oral administration of ginsenoside Rb1 from Panax ginseng. J. Pharm. Pharmacol. 1998, 50, 1155. (21) Hasegawa, H.; Sung, J. H.; Benno, Y. Role of human intestinal Prevotella oris in hydrolyzing ginseng saponins. Planta Med. 1997, 63, 436−440. (22) Takino, Y. Studies on the pharmacodynamics of ginsenosideRg1, -Rb1 and -Rb2 in rats. Yakugaku Zasshi 1994, 114, 550−564. (23) Hasegawa, H. Proof of the mysterious efficacy of ginseng. Basic and clinical trials: Metabolic activation of ginsenoside: Deglycosylation by intestinal bacteria and esterification with fatty acid. J. Pharmacol. Sci. (Tokyo) 2004, 95, 153−157. (24) Joo, K. M.; Lee, J. H.; Jeon, H. Y.; Park, C. W.; Hong, D. K.; Jeong, H. J.; Lee, S. J.; Lee, S. Y.; Lim, K. M. Pharmacokinetic study of ginsenoside Re with pure ginsenoside Re and ginseng berry extracts in mouse using ultra performance liquid chromatography/mass spectrometric method. J. Pharm. Biomed. Anal. 2010, 51 (1), 278−283. (25) Xie, H. T.; Wang, G. J.; Lv, H.; Sun, R. W.; Jiang, X. L.; Li, H.; Wang, W.; Huang, C. R.; Xu, M. J. Development of a HPLC-MS assay for ginsenoside Rh2, a new anti-tumor substance from natural product and its pharacokinetic study in dogs. Eur. J. Drug Metab. Pharmacokinet. 2005, 30 (1−2), 63−67. (26) Xie, H. T.; Wang, G. J.; Sun, J. G.; Tucker, I.; Zhao, X. C.; Xie, Y. Y.; Li, H.; Jiang, X. L.; Wang, R.; Xu, M. J.; Wang, W. High performance liquid chromatographic−mass spectrometric determination of ginsenoside Rg3 and its metabolites in rat plasma using solidphase extraction for pharmacokinetic studies. J Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2005, 818 (2), 167−173. (27) Dong, H.; Bai, L. P.; Wong, V. K.; Zhou, H.; Wang, J. R.; Liu, Y.; Jiang, Z. H.; Liu, L. The in vitro structure-related anti-cancer activity of ginsenosides and their derivatives. Molecules (Basel, Switzerland) 2011, 16 (12), 10619−10630. (28) Wakabayashi, C.; Hasegawa, H.; Murata, J.; Saiki, I. In vivo antimetastatic action of ginseng protopanaxadiol saponins is based on

minor metabolites derived from hydration and oxygenation may also have potential activity that needs to be further explored.

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ASSOCIATED CONTENT

S Supporting Information *

Characterization of the four groups of major metabolites. This material is available free of charge via the Internet at http:// pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Authors

*For Z.-H.J.: phone, +853-8897 2777; fax, +853-2882 5886; Email, [email protected]; address, State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology Taipa, Macau 00853, China. *For L.L.: phone, +853-8897 2077; fax, +853-2882 7222; Email, [email protected]. Funding

This project is sponsored by Macao Science and Technology Development Fund (066/2011/A3 to W.J.R.). Notes

The authors declare no competing financial interest.

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REFERENCES

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