Characterization of Oxygenated Metabolites of Ginsenoside Rb1 in

Article pubs.acs.org/JAFC

Characterization of Oxygenated Metabolites of Ginsenoside Rb1 in Plasma and Urine of Rat Jing-Rong Wang,†,§ Lee-Fong Yau,† Tian-Tian Tong,† Qi-Tong Feng,† Li-Ping Bai,†,§ Jing Ma,§ 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, China § School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China # Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, Texas, United States S Supporting Information *

ABSTRACT: Oxygenated metabolites have been suggested as the major circulating metabolites of ginsenosides. In the current study, 10 oxygenated metabolites of ginsenoside Rb1 in plasma and urine of rat following iv dose were characterized by comparison with chemically synthesized authentic compounds as quinquenoside L16 (M1 and M2), notoginsenoside A (M3), ginsenoside V (M4 and M7), epoxyginsenoside Rb1 (M5 and M9), notoginsenoside K (M6), and notoginsenoside C (M8 and M10), 9 of which were detected as in vivo metabolites for the first time. After oral administration of ginsenoside Rb1, M3, M4, and M7 were observed as major circulating metabolites and presented in the bloodstream of rat for 24 h. Characterization of the exact chemical structures of these circulating metabolites could contribute greatly to our understanding of chemical exposure of ginsenosides after consumption of ginseng products and provide valuable information for explaining multiple bioactivities of ginseng products. KEYWORDS: ginseng, ginsenoside Rb1, oxygenation, metabolites

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established.12,13 For example, compound K (C-K) is one of the major intestinal bacterial metabolites of PPD-type ginsenosides, and it was reported that the metabolism of Rb1 to C-K was dependent on different kinds of intestinal bacteria. Eubacterium sp., Streptococcus sp., and Bifidobacterium sp. metabolized Rb1 to C-K via ginsenoside Rd rather than gypenoside XVII, whereas Fusobacterium K-60 metabolized Rb1 to C-K via gypenoside XVII.14 However, very limited information on the metabolites reaching the circulation system is available as most of the gastrointestine-derived metabolites were poorly absorbed due to the involvement of efflux transport.15,16 On the other hand, the existence of hitherto unidentified metabolites in circulation have been indicated as exemplified by the radioisotope assay, which revealed 3 times higher serum radioactivity of Rb2 and its derivatives than Rb2 level determined by HPLC.17 Recent studies have shown that absorption of intact ginsenosides might not be as low as suggested previously, especially when ginseng extract was taken.18 Moreover, xueshuantong or xuesaitong injection, a preparation that is made of the total ginsenosides from notoginseng, has been widely used in China for the treatment of cerebral vascular disease. Primary ginsenosides (such as Rb1, Rg1, and Re) in the injection enter the circulation system without undergoing gastrointestinal decomposition, strongly indicating potential

INTRODUCTION Ginseng, the roots and rhizomes of Panax genus, for example, Panax ginseng (Asian ginseng) and Panax quinquefolius (American ginseng), has been used for thousands of years. It still occupies a prominent position in the herbal medicine market and is considered as the most widely taken health product in the world. It is estimated that more than six million Americans are regularly consuming ginseng herbs and their products.1 The root of Panax notoginseng (Burk.) F. H. Chen, commonly referred to as notoginseng and “sanqi”, is another well-known medicinal herb of Panax genus in traditional Chinese medicine. In addition to the medicinal usage, ginseng and American ginseng, as well as notoginseng, are also widely consumed as functional foods in the forms of tea, powder, capsule, and functional foods. In the United States, a variety of ginseng products are available as over the counter (OTC) dietary supplements in the health food market.2,3 Ginsenosides, a special group of triterpenoid saponins found nearly exclusively in Panax species, have long been believed to be responsible for most of the observed activities of ginseng and notoginseng.4−8 As a group of the most attractive chemical entities, the metabolism and absorption of ginsenosides have gained momentum as a key area for explaining the wide spectrum of therapeutic effects of ginsenosides, for example, anticancer and chemoprevention effects,9−11 and their contribution to the beneficial effects of ginseng products. Extensive biotransformation of ginsenosides in the gastrointestinal tract has been well appreciated, and metabolic pathways via stepwise cleavage of sugar moieties in ginsenosides have been well © 2015 American Chemical Society

Received: Revised: Accepted: Published: 2689

October 18, 2014 March 3, 2015 March 3, 2015 March 3, 2015 DOI: 10.1021/acs.jafc.5b00710 J. Agric. Food Chem. 2015, 63, 2689−2700

Article

Journal of Agricultural and Food Chemistry

Figure 1. Extracted compound chromatograms (ECC) of metabolites in rat plasma and urine after iv injection of Rb1 at a dosage of 20 mg/kg: (A) metabolites in plasma sample collected at 5 min after iv injection; (B) metabolites in urine samples collected during 3−6 h after iv dose of Rb1. purchased from Chengdu MUST Bio-Technology Co., Ltd. (Chengdu, China). Rose Bengal, triphenylphosphine, Shi Epoxidation Diketal Catalyst (ketone), OXONE, EDTA-Na, and sodium bicarbonate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Liver microsomes, NADPH Regenerating System Solution A (containing 31 mM NADP+, 66 mM glucose-6-phosphate, and 66 mM MgCl2) and Solution B (containing 40 U/mL glucose-6-phosphate dehydrogenase in 5 mM sodium citrate) were purchased from BD Biosciences (San Jose, CA, USA). Saline was purchased from Baxter (Deerfield, IL, USA). HPLC grade methanol (MeOH) and acetonitrile (ACN) were purchased from Anaqua Chemicals Supply Inc., Ltd. (Houston, TX, USA), and distilled water was purified by Milli-Q system (Millipore, Billerica, MA, USA). All other chemicals were of analytical reagent grade. Animals. Male SD rats (n = 2 for each route of administration) weighing 200−220 g were purchased from the Laboratory Animal Services Center, Chinese University of Hong Kong. All animals were acclimated for more than 1 week under a 12 h light/12 h dark cycle at a room temperature of 22 ± 1 °C. Animal care and treatment procedures were in accordance with the Institutional Guidelines and Animal Ordinance (Department of Health, Hong Kong Special Administrative Region) and approved by the Committee on the Use of Human and Animal Subjects in Teaching and Research of the Hong Kong Baptist University. The rats were fasted overnight but with access to water before the experiment. UHPLC/TOF MS Analysis of Metabolites. Metabolites in the biological samples of both in vivo and in vitro metabolism experiments were analyzed by using an Agilent 1290 Infinity ultrahigh-performance liquid chromatography (UHPLC) system coupled to an Agilent 6230 accurate-mass time-of-flight (TOF) mass spectrometer (MS) (Agilent, Santa Clara, CA, USA). MS/MS experiments were carried out on an Agilent 1290 Infinity UHPLC system coupled with an Agilent 6550 quadrupole-time-of-flight (Q-TOF) MS. The chromatography was performed on a Waters Acquity BEH C18 column (2.1 × 100 mm, 1.7 μm). The mobile phase consisted of 0.1%

pharmacological effects of the primary ginsenosides as well as their relevant metabolites. However, metabolism of the intact ginsenosides following intravenous administration remains unknown. Consequently, a huge gap between the considerable systemic effects of primary ginsenosides and metabolites reaching circulation system still exist. In addition to the well-known deglycosylation and dehydration pathways, ginsenosides have been increasingly reported to undergo oxidation.19−25 Oxygenated metabolites of both intact and deglycosylated ginsenosides have been observed as circulating metabolites.19,20,22,26,27 Recently, structures of oxygenated metabolites formed in liver microsomes of secondary ginsenosides, that is, Rh2,25 PPD,28 Rh1, and Rg2,29 have been elucidated. However, primary ginsenosides appeared to be metabolized via pathways distinct from that of secondary ginsenosides as they were not metabolized in microsomes. Structures of resulting oxygenated metabolites of primary ginsenosides were still not fully characterized due to technique difficulties, that is, a limitation in determining the exact oxygenated site solely by mass spectrometry, and difficulties in obtaining metabolites in adequate amounts for NMR measurements. As aforementioned, we herein carried out an extensive in vivo metabolite profiling of ginsenoside Rb1, the representative PPD-type primary ginsenoside in ginseng, American ginseng, and notoginseng, with a focus on the characterization of its oxygenated metabolites in rat plasma and urine.

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MATERIALS AND METHODS

Chemicals and Materials. Ginsenoside Rd and 20(S)-Rg3 (purity ≥ 98%) were purchased from Chengdu Scholar Biotech Co., Ltd. (Chengdu, China). Ginsenoside Rb1 and F2 (purity ≥ 98%) were 2690

DOI: 10.1021/acs.jafc.5b00710 J. Agric. Food Chem. 2015, 63, 2689−2700

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Figure 2. continued

2691


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Figure 2. High-resolution MS/MS of urinary M1−M10 obtained on Q-TOF MS. formic acid in Milli-Q water (A) and ACN containing 0.1% formic acid (B). The optimized UPLC elution condition as follows was employed: from 10 to 40% B at 0−23 min, from 40 to 85% B at 23−27 min, from 85 to 100% B at 27−29 min, and finally maintained at 100% B for 3 min at a flow rate of 0.35 mL/min. The injection volume was 5 μL. The ESI-MS was performed in the negative mode, and the source parameters were as follows: dry gas (N2) temperature and flow rate were 325 °C and 11 L/min, respectively; sheath gas (N2) temperature and flow rate were 350 °C and 11 L/min, respectively; nebulizer, 40 psi; fragmentor voltage, 175 V; skimmer voltage, 65 V. The acquisition mass range was m/z 100−1700, and the MS scan rate was 2 spectra/s. A reference solution was nebulized for continuous calibration in the negative mode using the reference masses of m/z 112.9856 and 966.0007. The targeted MS/MS collision energy (CE) was set at three different values from 20 to 60 eV. All MS and MS/MS data were processed using Agilent MassHunter Qualitative Analysis B.06.00 software. In Vivo Metabolism of Ginsenoside Rb1 Following Intravenous Injection and Oral Administration in Rat. Ginsenoside Rb1 was first dissolved in saline (8 mg/mL), and then a single intravenous (iv) dose of Rb1 solution (20 mg/kg) was given through the caudal vein. The blood (0.2 mL) was withdrawn via medial canthus of the eye at 0, 5, 15, and 30 min and 1, 2, 3, 4, 5, 6, 7, 24, and 48 h postadministration. One hundred microliters of plasma was collected by centrifuging the blood at 4000 rpm for 5 min, and then 200 μL of ACN was added for protein precipitation and ginsenoside extraction. After centrifugation at 14000 rpm for 5 min, the supernatant was transferred into a new tube and dried by speed vacuum. The residue was reconstituted with 100 μL of 50% MeOH. Each sample was

centrifuged at 14000 rpm for 5 min before UPLC/TOF MS analysis. For oral administration, Rb1 was dissolved in water (2.5 mg/mL) and given at a dose of 50 mg/kg. The blood (0.2 mL) was withdrawn via medial canthus of the eye at 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 8.5, 9, 9.5, 10, 24, 27, and 34 h postadministration. Urine samples were collected at time intervals of 0−3, 3−7, 7−10, 10−24, and 24−34 h. For analysis, 1 mL of urine was extracted with 0.5 mL of water-saturated n-BuOH three times. The n-BuOH layers were then combined and dried by speed vacuum. The resulting residue was reconstituted in 100 μL of 50% MeOH and filtered through a 0.2 μm filter. Chemical Synthesis of Oxygenated Metabolites. Photosensitized oxygenation was carried out to synthesize the oxygenated products following our protocol described previously.23 Briefly, a solution of ginsenoside Rb1 (150 mg) and Rose Bengal (150 mg) in 120 mL of MeOH was stirred and irradiated with a 400 W lamp under an oxygen atmosphere at room temperature for 6 h to synthesize crude dioxygenated Rb1 (Rb1 + 2O). Triphenylphosphine (35 mg) was further added to the reaction solution and was stirred at room temperature for 2 h for the production of crude mono-oxygenated Rb1 (Rb1 + O). Both solutions were evaporated to a small volume under reduced pressure and then directly subjected to a reverse phase preparative column, respectively (isocatic elution with 35% ACN at the flow rate of 2.5 mL/min) to give individual mono-oxygenated Rb1 (Rb1 + O (I), Rb1 + O (II), and Rb1 + O (III)) and individual dioxygenated Rb1 (Rb1 + 2O (I), Rb1 + 2O (II), and Rb1 + 2O (III)). Epoxidation was performed following the procedures described in the literature with minor modifications in our laboratory.23 Briefly, a solution of ginsenoside Rb1 (100 mg) in ACN (10 mL) was mixed with an aqueous EDTA-Na solution (0.4 μM, 10 mL). The mixed 2692


1141.6011

1123.5906

1121.5749 961.5378 795.4536

961.5378 1105.5800 945.5428 783.4900 783.4900

12.1

13.3

13.9

14.1

14.2 14.3

14.5

14.6

15.2

14.9 14.9 16.1

16.4 18.1 21.5 25.1 25.7

M4 (Rb1 + O)

M5 (Rb1 + O)

M6 (Rb1 + 2O) M7 (Rb1 + O)

M8 (Rb1 + 2O)

M9 (Rb1 + O)

M10 (Rb1 + 2O)

M11 (Rb1+O-2H) M12 (Rd + O) M13 (Rk1/Rg5 + 2O − 2H) M14 (Rd + O) M15 (Rb1 − 2H) M16 (Rd) M17 (F2) M18 [20(S)-Rg3]

1139.5855

1123.5906

1139.5855

1139.5855 1123.5906

1123.5906

1123.5906

1107.5957 1141.6011

theor mass (m/z)

18.8 10.9

RT (min)

ginsenoside Rb1 M1 (Rb1 + O + H2O) M2 (Rb1 + O + H2O) M3 (Rb1 + O)

metabolite

2693

961.5369 1105.5805 945.5426 783.4903 783.4899

1121.577 961.5373 795.453

1139.5887

1123.5937

1139.5839

1139.5863 1123.5939

1123.5923

1123.5916

1123.5912

1141.6026

1107.5984 1141.6005

measured mass (m/z)

C54H90O24 C48H82O19 C42H68O14 C48H82O19 C54H90O23 C48H82O18 C42H72O13 C42H72O13

−0.94 0.45 −0.21 0.38 −0.13

C54H92O25

1.87 −0.52 −0.75

2.81

C54H92O24

C54H92O25

−1.40 2.76

C54H92O25 C54H92O24

C54H92O24

C54H92O24

C54H92O24

C54H94O25

C54H92O23 C54H94O25

molecular formula

0.70 2.94

1.51

0.89

0.53

1.31

2.44 −0.53

error (ppm) −

CID (m/z)

799.4733 943.5252 783.4869 621.4317 621.4298

[M [M [M [M [M

− − − − − Glc Glc Glc Glc Glc

− − − − −

H]−, H]−, H]−, H]−, H]− 637.4408 781.4723 621.4342 459.3831

[M [M [M [M

− − − −

2Glc 2Glc 2Glc 2Glc

− − − −

H]− H]−, 619.4136 [M − 3Glc − H]−, 457.3683 [M − 4Glc − H]− H]−, 459.3834 [M − 3Glc − H]− H]−

945.5415 [M − Glc − H] , 783.4899 [M − 2Glc − H] , 621.4332 [M − 3Glc − H]−, 459.3839 [M − 4Glc − H]− 979.5511 [M − Glc − H]−, 817.4891 [M − 2Glc − H]−, 799.4822 [M − 2Glc − H2O − H]−, 655.4423 [M − 3Glc − H]−, 493.3872 [M − 4Glc − H]− 979.5415 [M − Glc − H]−, 817.4904 [M − 2Glc − H]−, 799.4829 [M − 2Glc − H2O − H]−, 655.4440 [M − 3Glc − H]−, 493.3920 [M − 4Glc − H]− 961.5493 [M − Glc − H]−, 799.4850 [M − 2Glc − H]−, 781.4752 [M − 2Glc − H2O − H]−, 637.4320 [M − 3Glc − H]−, 619.4202 [M − 3Glc − H2O − H]−, 475.3919 [M − 4Glc − H]− 961.5373 [M − Glc − H]−, 799.4833 [M − 2Glc − H]−, 781.4724 [M − 2Glc − H2O − H]−, 637.4305 [M − 3Glc − H]−, 619.4211 [M − 3Glc − H2O − H]−, 475.3794 [M − 4Glc − H]− 961.5351 [M − Glc − H]−, 799.4841 [M − 2Glc − H]−, 781.4734 [M − 2Glc − H2O − H]−, 637.4296 [M − 3Glc − H]−, 475.3786 [M − 4Glc − H]− 977.5209 [M − Glc − H]−, 815.4768 [M − 2Glc − H]−, 797.4663 [M − 2Glc − H2O − H]−, 635.4172 [M − 3Glc − H2O − H]− 961.5379 [M − Glc − H]−, 799.4850 [M − 2Glc − H]−, 781.4750 [M − 2Glc − H2O − H]−, 637.4334 [M − 3Glc − H]−, 619.4216 [M − 3Glc − H2O − H]−, 475.3799 [M − 4Glc − H]− 977.5268 [M − Glc − H]−, 815.4817 [M − 2Glc − H]−, 797.4668 [M − 2Glc − H2O − H]−, 635.4147 [M − 3Glc − H2O − H]−, 473.3615 [M − 4Glc − H2O − H]− 961.5350 [M − Glc − H]−, 799.4851 [M − 2Glc − H]−, 781.4719 [M − 2Glc − H2O − H]−, 637.4302 [M − 3Glc − H]−, 619.4230 [M − 3Glc − H2O − H]−, 475.3784 [M − 4Glc − H]− 977.5224 [M − Glc − H]−, 815.4669 [M − 2Glc − H]−, 797.4646 [M − 2Glc − H2O − H]−, 635.4163 [M − 3Glc − H2O − H]−, 473.3612 [M − 4Glc − H2O − H]− 959.5001 [M − Glc − H]−, 797.4634 [M − 2Glc − H]−, 779.4618 [M − 2Glc − H2O − H]−, 635.4229 [M − 3Glc − H]− 799.4759 [M − Glc − H]−, 781.4677 [M − Glc − H2O − H]−, 637.5666 [M − 2Glc − H]− 633.4008 [M − Glc − H]−, 615.3908 [M − Glc − H2O − H]−, 471.3461 [M − 2Glc − H]−, 453.3372 [M − 2Glc − H2O − H]−

−

Table 1. Characterized Metabolites in Rat Urine Following Intravenous Injection of Rb1 at a Dosage of 20 mg/kg ([M − H]− as Precursor Ion)

Journal of Agricultural and Food Chemistry Article


Article

Journal of Agricultural and Food Chemistry Scheme 1. Chemical Synthesis of Mono- and Dioxygenated Derivatives of Ginsenoside Rb1

solution was cooled to 0 °C, and then an epoxidation dibetal catalyst solution (ketone, 15 mM in ACN) and a mixture of sodium bicarbonate (67.5 mM) and OXONE (45 mM) were added. The homogeneous solution was stirred for 2 h. Because the epoxide was quite unstable and easily changed to diol (Rb1 + O + H2O) as a byproduct, a mixture of Rb1 epoxides (Rb1-Epo) and diols (Rb1 + O + H2O) appeared in the reaction solution. The reaction solution was reduced in volume by speed vacuum under low temperature and then was directly subjected to a reverse phase preparative column (gradient elution with 10−35% ACN at the flow rate of 2.5 mL/min) to afford individual Rb1 epoxides (Rb1-Epo (I) and Rb1-Epo (II)) and individual Rb1 diols (Rb1 + O + H2O (I) and Rb1 + O + H2O (II)). Chemical structures of the synthesized metabolites were characterized on the basis of the elucidation of NMR data. 1H and 13C NMR data were measured on a Bruker Ascend 600 NMR spectrometer (600 MHz for 1H NMR and 150 MHz for 13C NMR). In Vitro Metabolism of Ginsenoside Rb1. Metabolism of ginsenoside Rb1 in liver microsomes and S9 fraction was carried out initially by dissolving Rb1 in potassium phosphate buffer (pH 7.4), followed by the addition of NADPH Regenerating System Solutions A and B. After prewarming at 37 °C for 5 min, liver microsomes or S9 fraction was added and incubated at 37 °C for 2 h. The reaction was quenched by adding 2-fold ACN for protein precipitation and ginsenoside extraction. After centrifugation at 14000 rpm for 5 min, the supernatant was collected for analysis. Ginsenoside Rb1 was spiked in fresh blood, red blood cells, and plasma, respectively. After incubation at 37 °C for 2 h, 100 μL of H2O was added in the red blood cell sample and incubated at 37 °C for 5 min, whereas plasma was collected from whole blood by centrifugation at 4000 rpm for 5 min. Two-fold ACN was added in the cell lysed sample or plasma for protein precipitation and ginsenoside extraction. After centrifugation at 14000 rpm for 5 min, the supernatant was collected for analysis.

plasma and urine following iv administration were examined first. All possible metabolites were screened and compared with the profiles of the blank plasma and urine samples collected before administration. As a result, the metabolite profile in plasma samples was found to be generally consistent with that of urine samples (Figure 1A, B), but metabolites in urine samples appeared to be more abundant and diverse (Figure 1B) and therefore were characterized first. As shown in Figure 1, in total 18 metabolites (M1−M18) appeared in urine after dosing, among which three groups of peaks corresponding to oxygenated metabolites could be clearly observed at retention times smaller than that of Rb1 (chromatograms of blank samples are given in the Supporting Information). The first group of the peaks (M1 and M2) (tR of 10.5− 12.5 min) gave [M − H]− ion peaks at m/z exactly 34 Da more than that of Rb1, indicating introduction of an O and an H2O into the molecule of Rb1, which was confirmed by the molecular formula derived from accurate mass. The MS/MS spectra of M1 and M2 showed fragment ions corresponding to the sequential loss of glucose moieties (Figure 2; Table 1). The product ion derived from the loss of all sugar moieties was observed at m/z 493.3872, which was 34 Da greater than corresponding product ion of Rb1 at m/z 459.3839, indicating that the additional O and H2O were located on the aglycone moiety. The second group of metabolites (tR of 13.0−15.0 min) (M3, M4, M5, M7, and M9) gave [M − H]− ions that can be assigned as Rb1 + O on the basis of the accurate mass and isotope abundance, showing that these five metabolites were mono-oxygenated derivatives of Rb1. Targeted MS/MS experiments of these metabolites gave fragment ions derived from sequential cleavage of glycosidic bonds (Figure 2; Table 1), indicating the introduction of an O atom into the aglycone moiety of these metabolites. The last group of peaks (tR of 14.0−15.5 min) (M6, M8, and M10) was assigned as dioxygenated metabolites of Rb1 on the basis of accurate mass and isotope cluster information. MS/MS experiment revealed that the dioxygenation occurred at the aglycone

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RESULTS Characterization of Oxygenated Metabolites of Ginsenoside Rb1 in Rat Urine and Plasma after Intravenous Administration. It has been suggested that oxygenated metabolites of ginsenosides were majorly derived from tissue oxidation.27 Therefore, oxygenated metabolites of Rb1 in 2694


Article

Journal of Agricultural and Food Chemistry Scheme 2. Chemical Synthesis of Rb1 Epoxides and Corresponding Diols

moiety (Figure 2; Table 1). In all of the high-resolution MS/MS spectra of M1−M10 obtained on Q-TOF MS, the sequential fragment resulting from the loss of sugar could be clearly observed (Figure 2). Although all above oxygenations were suggested to occur on the aglycone part by MS/MS experiments, further structural information related to the exact oxygenation site cannot be obtained solely on the basis of MS/MS experiment. Therefore, possible oxygenated metabolites were chemically synthesized for further characterization. Taking into consideration the chemical characteristic of Rb1, as well as the possible oxygenation pathway that may lead to the incorporation of an oxygen atom into the molecule, we speculated that Rb1 may undergo initial oxygenation via either epoxidation or hydroxylation along with displacement of the double bond of the side chain. Therefore, we synthesized epoxides of Rb1 and photosensitized Rb1 by using Rb1 as starting material (Schemes 1 and 2). Chemical structures of these synthesized metabolites were characterized on the basis of spectroscopic data (Supporting Information). They were used as authentic standards for the identification of the above-mentioned oxygenated metabolites (Figure 3). The synthesized compounds were then employed as standards for the identification of the oxygenated metabolites, which was implemented by comparing retention time, highresolution MS, and MS/MS pattern of the metabolites with those of synthesized standards. Consequently, M1 and M2 were identified as a pair of enantiomers (24α- and 24β-forms) of 3-O-[β-D-glucopyranosyl (1→2)-β-D-glucopyranosyl]-20-O[β-D-glucopyranosyl (1→6)-β-D-glucopyranosyl] 3β,12β,20(S),24,25-pentahydroxy-dammarane (quinquenoside L16).30 The three major mono-oxygenated metabolites (M3, M4, and M7) were identified as 24- and 25-hydroxyl derivatives of Rb1, that is, 3-O-[β-D-glucopyranosyl (1→2)-β-D-glucopyranosyl]20-O-[β- D -glucopyranosyl (1→6)-β- D -glucopyranosyl] 3β,12β,20(S),25-tetrahydroxy-dammar-23-ene (notoginsenoside A) (M3),31 3-O-[β-D-glucopyranosyl (1→2)-β-D-glucopyranosyl]-20-O-[β-D-glucopyranosyl (1→6)-β-D-glucopyranosyl] 3β,12β,20(S)-trihydroxy-24ξ-hydroxydammar-25-ene (ginsenoside V) (M4 and M7),32 whereas the two minor monooxygenated metabolites (M5 and M9) were characterized to be epoxyginsenoside Rb1 (24α- and 24β-forms).33,34 The three dioxygenated metabolites (M6, M8, and M10) were identified as 24- and 25-hydroperoxides of Rb1, that is, 3-O-[β-D-glucopyranosyl (1→2)-β-D-glucopyranosyl]-20-O-[β-D-glucopyranosyl (1→6)-β-D-glucopyranosyl] 3β,12β,20(S)-trihydroxy-25-hydroperoxydammar-23-ene (notoginsenoside K) (M6)35 and 3-O[β-D-glucopyranosyl (1→2)-β-D-glucopyranosyl]-20-O-[β-Dglucopyranosyl (1→6)-β-D-glucopyranosyl] 3β,12β,20(S)trihydroxy-24ξ-hydroperoxydammar-25-ene (notoginsenoside C)

Figure 3. Characterization of oxygenated urinary metabolites M1− M10 (A−C, upper panels) by comparison with synthesized authentic metabolites (A−C, lower panels). 2695


Article


Figure 4. Proposed oxygenation metabolism pathway of ginsenoside Rb1 in vivo.

(M8 and M10).31 A metabolism pathway of the major oxygenated metabolites is proposed in Figure 4. In addition to the major oxygenated metabolites, another eight metabolites (M11−M18) were observed in rat urine after iv administration of Rb1 (Figure 1; Table 1). On the basis of the molecular formula derived from accurate mass, M11 was assigned as dehydrogenation product of mono-oxygenated Rb1 (Rb1 + O − 2H). M12 and M14 were tentatively identified as mono-oxygenated Rd (Rd + O). M13 was assigned as Rk1/Rg5 + 2O − 2H. M15 was identified to be the dehydrogenated derivative of Rb1 (Rb1 − 2H), whereas M16, M17, and M18 were characterized as Rd, F2, and 20(S)-Rg3 respectively, all of which were deglycosylated products of Rb1. Characterization of M16−M18 was further confirmed by comparison with authentic ginsenosides, suggesting that deglycosylation is another metabolic pathway of primary ginsenosides in the circulating system (Table 1). In plasma samples, metabolites M1 and M2, as well as five mono-oxygenated metabolites (M3, M4, M5, M7, and M9), can be clearly observed. Dehydrogenated metabolites (M15) and deglycosylated metabolites (M16, M17, and M18) also appeared in the bloodstream. However, three dioxygenated metabolites (M6, M8, and M10) together with oxygenated metabolites of secondary ginsenoside, that is, M12 and M14 (Rd + O) and M13 (Rk1/Rg5 + 2O − 2H), were absent in plasma samples. Because these urinary metabolites possessed

increased polarity relative to their parent ginsenosides, their absence in plasma might be due to fast elimination through kidney clearance. By contrast, the relative levels of the deglycosylated metabolites (M16−M18) in plasma were obviously higher than those in urine, implying that these metabolites were excreted into urine very slowly and thereby accumulated in the bloodstream. Metabolite Profile in Rat Urine and Plasma after Oral Administration of Rb1. After oral administration of Rb1, metabolites in rat plasma and urine were characterized by using a similar approach as that for the iv dose. Metabolites in both plasma and urine after oral administration were significantly less than that observed for iv administration. In plasma, nine metabolites in total were detected (Figure 5A), including three mono-oxygenated metabolites (M3, M4, and M7), two dehydrogenated metabolites (M11 and M15), and two deglycosylated metabolites (M16 and M18), as well as oxygenated Rg3 (M19). Mono-oxygenated Rb1, M3, M4, and M7 were found to be the major metabolites of Rb1 appearing in the circulating system. The existence of these metabolites in blank plasma and urine samples was examined, and the results suggested that these metabolites were derived from the metabolism of ginsenosides rather than interferences preexisting in plasma or urine (Supporting Information). To survey the systemic availability of these metabolites, rat plasma samples collected at different time points were 2696


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Figure 5. Ginsenoside Rb1 and its metabolites identified in plasma following oral administration: (A, B) extracted compound chromatograms (ECC) of metabolites identified in plasma collected at 0.5 and 1.5 h after oral dose of Rb1; (C) appearance of metabolites in rat plasma at different time intervals after oral dose of Rb1.

metabolites, M1, M2, and M9, were mainly measured in urine samples within 7 h after oral administration, whereas M19 appeared in urine only within 3 h after oral dose. In Vitro Metabolism of Rb1 in Liver Microsomes, S9 Fraction, and Rat Blood. To get information about the metabolism site of the oxygenated metabolites, in vitro metabolism of Rb1 in liver microsomes and blood was investigated. Hepatic metabolism is the leading drug disposition pathway because liver is the major site of drug metabolism in which a complex array of phase I, phase II, and phase III transporter systems function in the systemic clearance of xenobiotics. Several studies have revealed the involvement of P450-mediated metabolism in the elimination of secondary ginsenosides and proposed oxygenation as the major metabolic pathway.19,20,22,36 Therefore, biotransformation of Rb1 in liver microsomes and S9 fraction was examined. However, neither oxygenated products nor other metabolites were observed during the incubation duration of 2 h. The results were consistent with that observed for the primary PPT-type ginsenosides.29 Similarly, no metabolites of Rb1 were detected

examined for all identified metabolites. As shown in Figure 5C, Rb1, M3, M15, M16, and M18 were found to be continuously present in the bloodstream for >24 h. However, M4, M7, M11, and M12 appeared in the plasma at intervals of 0−8 h and after 24 h, whereas the oral administration-specific metabolite M19 was measured within only 1.5 h after administration. The time duration for the occurrence of the metabolites provided clues for their metabolism site. For instance, the metabolites detected at the early stage, for example, oxygenated Rd (M12), might result from oxidation of deglycosylated metabolites that derived from gastric acid decomposition. By contrast, the oxygenated Rd observed at the late stage might result from tissue oxidation of intestinal-decomposed Rb1. The plasma metabolites were also excreted into urine (Figure 6A). Twelve metabolites were detected in urine, among which M1, M2, and M9 were not found in plasma. The appearance duration of these metabolites in urine showed certain differences. Seven abundant metabolites, M3, M4, M7, M11, M12, M15, M16, and M18, were detected in urine samples for a long duration (up to 24 h). Less abundant 2697


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Figure 6. Ginsenoside Rb1 and its metabolites identified in urine following oral administration: (A, B) extracted compound chromatograms (ECC) of metabolites identified in urine samples collected at 0−3 and 3−7 h after oral dose of Rb1; (C) appearance of metabolites in rat urine at different time intervals after oral dose of Rb1.

By using such a chemical synthesis approach, we confirmed the generation of two 24,25-epoxides (M5 and M9) of primary ginsenoside as circulating metabolites in plasma and urine. Oxidation of the olefin bond to form epoxide is a well-known oxidation reaction catalyzed by CYP and therefore was proposed to be an important oxidation pathway of ginsenosides in several previous studies.25,28,29 However, as epoxides are quite reactive due to ring strain, their rearranged products, 20,24-epoxides, are usually observed as the final oxygenated metabolites. Alternatively, the reactive epoxides were captured with nucleophile trapping agents such as GSH to confirm the formation of these bioactive metabolites, whereas the epoxides themselves were normally undetectable. Our study represents the first report of the direct detection of 24,25-epoxides of Rb1 (M5 and M9) as in vivo metabolites. Accordingly, the diols of Rb1 (M1 and M2) were identified unambiguously as the in vivo metabolites of Rb1 for the first time. Notably, after oral administration, mono-oxygenated metabolites (M3, M4, and M7) represented major metabolites of Rb1 reaching the circulation system and appearing in the bloodstream once Rb1 was absorbed. These oxygenated metabolites presented in plasma continuously for 8−24 h after orally taken. Two diol metabolites (M1 and M2) also appeared in urine samples as abundant metabolites. As the sequential metabolites of epoxides (M5 and M9), the appearance of M1 and M2 in urine suggested the formation of 24,25-epoxides of Rb1 in the circulation system. These results concomitantly indicated notable exposure of oxygenated metabolites of Rb1 after oral administration. The oxygenated metabolism of ginsenosides with additional hydroxyl or epoxy groups in the side chain may lead to changes in the bioactivity. It has been reported that notoginsenoside H,

when it was incubated in fresh blood, red blood cells, and plasma for 2 h, showing that Rb1 was quite stable in blood. These results suggested that oxygenated metabolites of Rb1 might result from tissue oxidation as previously suggested.27

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DISCUSSION Primary ginsenosides such as Rb1 and Rg1, although poorly absorbed after oral administration, were still demonstrated to be relatively abundant ginsenosides reaching the circulation system after oral administration of notoginseng extract, in comparison with secondary ginsenosides such as Rg3 and Rg2.27 In multiple-dose treatment, the primary ginsenosides were found to further accumulate substantially in the systemic circulation, resulting in a considerably higher plasma level.26 Metabolism of the primary ginsenosides after entering the bloodstream represented one of the essential issues for further evaluation of these intact ginsenosides. Tissue oxygenation has been suggested to be the major metabolism pathway of primary ginsenosides apart from deglycosylation and dehydration in the gastrointestinal tract.20,24 The resulting mono-oxygenated metabolites were further estimated to account for about 10−15% elimination of intravenously administered Rb1 in a single iv dose.27 These results collectively directed the necessity for the characterization of the chemical structures of oxygenated metabolites. To overcome the technique limitations for structural characterization of metabolites, that is, accumulating adequate amount of metabolites for NMR measurements, we utilized a chemical synthesis approach to obtain chemical standards and unambiguously identified five mono-oxygenated metabolites, three dioxygenated metabolites, and two diol metabolites of Rb1 in plasma and urine for the first time. 2698


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(4) Wang, J. R.; Yamasaki, Y.; Tanaka, T.; Kouno, I.; Jiang, Z. H. Dammarane-type triterpene saponins from the flowers of Panax notoginseng. Molecules 2009, 14, 2087−2094. (5) Wang, J. R.; Leung, C. Y.; Ho, H. M.; Chai, S.; Yau, L. F.; Zhao, Z. Z.; Jiang, Z. H. Quantitative comparison of ginsenosides and polyacetylenes in wild and cultivated American ginseng. Chem. Biodivers. 2010, 7, 975−983. (6) Xie, C. L.; Wang, W. W.; Xue, X. D.; Zhang, S. F.; Gan, J.; Liu, Z. G. A systematic review and meta-analysis of ginsenoside-Rg1 (G-Rg1) in experimental ischemic stroke. Sci. Rep. 2015, 5, 7790. (7) Zhang, Y.; Yu, L.; Cai, W.; Fan, S.; Feng, L.; Ji, G.; Huang, C. Protopanaxatriol, a novel PPARγ antagonist from Panax ginseng, alleviates steatosis in mice. Sci. Rep. 2014, 4, 7375. (8) Liu, J.-H.; Lee, C.-S.; Leung, K.-M.; Yan, Z.-K.; Shen, B.-H.; Zhao, Z.-Z.; Jiang, Z.-H. Quantification of two polyacetylenes in radix ginseng and roots of related Panax species using a gas chromatography-mass spectrometric method. J. Agric. Food Chem. 2007, 55, 8830−8835. (9) 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 2011, 16, 10619−10630. (10) Wong, V. K.; Cheung, S. S.; Li, T.; Jiang, Z. H.; Wang, J. R.; Dong, H.; Yi, X. Q.; Zhou, H.; Liu, L. Asian ginseng extract inhibits in vitro and in vivo growth of mouse lewis lung carcinoma via modulation of ERK-p53 and NF-κB signaling. J. Cell. Biochem. 2010, 111, 899− 910. (11) Kwok, H. H.; Guo, G. L.; Lau, J. K.; Cheng, Y. K.; Wang, J. R.; Jiang, Z. H.; Keung, M. H.; Mak, N. K.; Yue, P. Y.; Wong, R. N. Stereoisomers ginsenosides-20(S)-Rg(3) and -20(R)-Rg(3) differentially induce angiogenesis through peroxisome proliferator-activated receptor-γ. Biochem. Pharmacol. 2012, 83, 893−902. (12) 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 2005, 818, 167−173. (13) 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, 63−67. (14) Bae, E. A.; Park, S. Y.; Kim, D. H. Constitutive β-glucosidases hydrolyzing ginsenoside Rb1 and Rb2 from human intestinal bacteria. Biol. Pharm. Bull. 2000, 23, 1481−1485. (15) Yang, Z.; Wang, J. R.; Niu, T.; Gao, S.; Yin, T.; You, M.; Jiang, Z. H.; Hu, M. Inhibition of P-glycoprotein leads to improved oral bioavailability of compound K, an anticancer metabolite of red ginseng extract produced by gut microflora. Drug Metab. Dispos. 2012, 40, 1538−1544. (16) Yang, Z.; Gao, S.; Wang, J.; Yin, T.; Teng, Y.; Wu, B.; You, M.; Jiang, Z.; Hu, M. Enhancement of oral bioavailability of 20(S)ginsenoside Rh2 through improved understanding of its absorption and efflux mechanisms. Drug Metab. Dispos. 2011, 39, 1866−1872. (17) 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. 2004, 95, 153−157. (18) Li, X.; Wang, G.; Sun, J.; Hao, H.; Xiong, Y.; Yan, B.; Zheng, Y.; Sheng, L. Pharmacokinetic and absolute bioavailability study of total panax notoginsenoside, a typical multiple constituent traditional chinese medicine (TCM) in rats. Biol. Pharm. Bull. 2007, 30, 847−851. (19) Qian, T.; Cai, Z.; Wong, R. N.; Mak, N. K.; Jiang, Z. H. In vivo rat metabolism and pharmacokinetic studies of ginsenoside Rg3. J. Chromatogr., B 2005, 816, 223−232. (20) Qian, T.; Jiang, Z. H.; Cai, Z. High-performance liquid chromatography coupled with tandem mass spectrometry applied for metabolic study of ginsenoside Rb1 on rat. Anal. Biochem. 2006, 352, 87−96.

a saponin having a side chain the same as that of metabolite M3, exhibited immunological adjuvant activity by profoundly increasing the level of IgG in mice sensitized with ovalbumin. Rb1 epoxide is a patented apoptosis inhibitor and regeneration promoter,33 implicating its potential bioactivity. On the basis of the mass of the evidence, the mono-oxygenated metabolites of ginsenosides might contribute to the observed pharmacological activity and therapeutic effect of ginsenosides and ginseng product, which needs to be further explored. Collectively, the structures of 10 oxygenated metabolites in rat urine and plasma after iv administration of Rb1 were unambiguously characterized. These metabolites were further proved to be major metabolites of ginsenoside Rb1 appearing in the bloodstream after oral administration. As the major circulating metabolites of ginsenoside Rb1 after both intravenous injection and oral administration, these metabolites have great potential to be responsible for the observed pharmacological activity and therapeutic effect of ginsenosides. Therefore, exact characterization of the chemical structures of these circulating oxygenated metabolites would shed light on the clarification of possible toxic or pharmacologically active metabolites, thus providing valuable information for explaining the multiple pharmacological activities of ginseng products. The results were also informative for the design of the next generation of drugs to circumvent the undesired metabolic fate of ginsenosides. Moreover, clarification of metabolites reaching the circulation system will lay the foundation for the assessment of pharmacokinetics of ginsenosides and its metabolites. Corresponding results will in turn provide important clues pertaining to various aspects of the disposition of ginsenosides, including first-pass metabolism, elimination half-life, and overall bioavailability.

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ASSOCIATED CONTENT

* Supporting Information S

Characterization of M1−M10. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(Z.-H.J.) E-mail: [email protected]. Phone: + 853-8897 2777. Fax: + 853-2882 5886; *(L.L.) E-mail: [email protected]. Phone: +853-8897 2077. Fax: +853-2882 7222. Funding

This project is sponsored by the Macao Science and Technology Development Fund (066/2011/A3 to J.-R.W.) and the National Institutes of Health, USA (AT005522-03). Notes

The authors declare no competing financial interest.

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Characterization of Oxygenated Metabolites of Ginsenoside Rb1 in

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