Determination of American ginseng saponins and their metabolites in human plasma, urine and feces samples by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry

https://doi.org/10.1016/j.jchromb.2016.02.008Get rights and content

Highlights

  • American ginseng saponins and their metabolites in human plasma, urine and feces after oral administration by LC-Q-TOF-MS.

  • The ginseng constituents and metabolites were characterized, including 5 metabolites in plasma, 10 in urine, and 26 in feces.

  • The levels of Rb1 and compound K were determined, and compound K had remarkably high level in all three kinds of biosamples.

  • Ginsenoside Rk1 and Rg5, Rk3 and Rh4, Rg6 and F4 were detected as the products of dehydration in human feces.

Abstract

American ginseng is a commonly consumed herbal medicine in the United States and other countries. Ginseng saponins are considered to be its active constituents. We have previously demonstrated in an in vitro experiment that human enteric microbiota metabolize ginseng parent compounds into their metabolites. In this study, we analyzed American ginseng saponins and their metabolites in human plasma, urine and feces samples by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). Six healthy male volunteers ingested 1 g of American ginseng twice a day for 7 days. On day 7, biological samples were obtained and pretreated with solid phase extraction. The ginseng constituents and their metabolites were characterized, including 5 ginseng metabolites in plasma, 10 in urine, and 26 in feces. For the plasma, urine and feces samples, the levels of ginsenoside Rb1 (a major parent compound) were 8.6, 56.8 and 57.7 ng/mL, respectively, and the levels of compound K (a major metabolite) were 58.4 ng/mL, 109.8 ng/mL and 10.06 μg/mL, respectively. It suggested that compound K had a remarkably high level in all three samples. Moreover, in human feces, ginsenoside Rk1 and Rg5, Rk3 and Rh4, Rg6 and F4 were detected as the products of dehydration. Further studies are needed to evaluate the pharmacological activities of the identified ginseng metabolites.

Introduction

American ginseng or Panax quinquefolius is a commonly consumed herbal medicine in the United States and other countries. Ginseng saponins are considered to be active constituents of this botanical [1]. Until now, nearly a hundred ginsenosides have been isolated and characterized from American ginseng, and many of them have shown pharmacological activities [2], [3]. Yet, compared with studies of other ginseng species, such as Panax ginseng and Panax notoginseng, analytical studies of American ginseng and its components are relatively limited [4], [5].

The chemical and pharmacological diversity of different ginseng constituents has been investigated. In studies of many parent compounds of ginseng, attention also has been given to their potential structure-activity relationship [6], [7], [8]. Reductionist methodology in ginseng research during in vitro screening has been applied primarily to the bioactivity of parent compounds [9], [10]. However, the bioavailability of ginseng compounds, an important consideration for their effects in vivo, has been overlooked.

Of the commercially available American ginseng products, nearly all are ingested orally. After oral intake, trillions of gut microbiota in the gastrointestinal tract may affect ginseng biotransformation [11]. Metabolic profiles of ginseng by enteric microbiota have been reported in animals [12], [13], and the actions of selected biotransformed metabolites have been elucidated [14]. For example, compound K, a major metabolite in the protopanaxadiols group, has a better bioactivity than its parent compound, ginsenoside Rb1 [15]. In an in vitro study we observed that human enteric microbiota metabolized ginseng parent compounds into 25 metabolites [16]. However, to date, the determination of ginseng metabolites in human biological samples has largely not been carried out. To link the health benefits of ginseng compounds to their effects, we sought to determine the profiles of ginseng and its metabolites after oral administration.

Based on our previous studies, liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) can be effectively used to characterize ginseng saponins and their metabolites [16]. This technique provides advanced structural information with high sensitivity, specificity, and versatility in characterizing complex metabolite profiles in matrix-based samples. With this method, all ginseng constituents, even at low concentrations, can be successfully detected.

In a previous study in humans, we analyzed a ginsenoside and its metabolism in plasma after a single oral dose of American ginseng using UPLC-TOF-MS [17]. It was indicated that the metabolite profile might not have been adequately revealed after a single dose regimen in the qualitative study. For the study reported here, we recruited six healthy volunteers to ingest American ginseng for 7 successive days. The 2 g daily ginseng used in this study was within the commonly used therapeutic dose range. Plasma, urine and feces samples were collected at the end of the 7 days and analyzed by LC-Q-TOF-MS. Special attention was paid to the determination of a major ginseng parent compound, ginsenoside Rb1, and its predominant metabolite, compound K.

Section snippets

Chemicals and reagents

Reference ginsenosides, including ginsenoside Rb1, Rb2, Rb3, Rc, Rd, Re, Rg1, F1, F2, pseudoginsenoside F11, gypenoside XVII, 20S-Rg2, 20R-Rg2, 20S-Rg3, 20R-Rg3, 20S-Rh1, 20R-Rh1, 20S-Rh2, 20R-Rh2, compound K, and protopanaxatriol were purchased from Jilin University (Changchun, China). Ginsenoside Rk3, Rh4, Rk1 and Rg5 were prepared in our laboratory by steaming transformation. The structures are shown in Fig. 1C, and were elucidated by 1H NMR, 13C NMR and MS. The internal standard (IS)

Analytical condition optimization

In this study, LC-Q-TOF-MS was used for the online structural characterization of American ginseng saponins and their metabolites in human plasma, urine and feces samples.

Considering the chemical complexity of the biological matrixes, the effects of chromatographic conditions, including the column, mobile phases and modifiers, were evaluated. It was shown that Zorbax Extend-C18 column was the most suitable for the separation of ginsenosides and the relevant metabolites. A solvent system of

Conclusion

In this study, we tested American ginseng saponins and their metabolites in plasma, urine and feces samples from six human subjects. A total of 5, 10, and 26 metabolites were detected in the plasma, urine and feces, respectively, using LC-Q-TOF-MS technology. Out of all the metabolites, compound K was found at the highest levels in these biological samples. It is likely that ginsenoside Rb1 was biotransformed into compound K via gastrointestinal microbiota. This biotransformation appears to

Acknowledgments

We thank Sally Kozlik for editing the manuscript. This work was supported in part by grants from the NIH/NCCAM AT004418 and AT005362, the Senior Talent Cultivation Program of Jiangsu University (15JDG069), the Priority Academic Program Development of Jiangsu Higher Education Institution (PAPD).

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