Accepted Manuscript Title: Metabolic profiling of Gynostemma pentaphyllum extract in rat serum, urine and faeces after oral administration Author: Dao-Jin Chen Hua-Gang Hu Shao-Fang Xing Ya-Jun Gao Si-Fan Xu Xiang-Lan Piao PII: DOI: Reference:
S1570-0232(14)00512-1 http://dx.doi.org/doi:10.1016/j.jchromb.2014.08.003 CHROMB 19065
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
17-3-2014 31-7-2014 3-8-2014
Please cite this article as: D.-J. Chen, H.-G. Hu, S.-F. Xing, Y.-J. Gao, S.-F. Xu, X.-L. Piao, Metabolic profiling of Gynostemma pentaphyllum extract in rat serum, urine and faeces after oral administration, Journal of Chromatography B (2014), http://dx.doi.org/10.1016/j.jchromb.2014.08.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Metabolic profiling of Gynostemma pentaphyllum extract in rat serum, urine and faeces after oral administration
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Dao-Jin Chena, Hua-Gang Hua, Shao-Fang Xinga, Ya-Jun Gaob, Si-Fan Xua, Xiang-Lan
Institute of Chinese Minority Traditional Medicine, Minzu University of China, Beijing
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Piaoa,*
100081, China
Department of Breast Disease Prevention Center, Haidian Maternal and Child Health
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b
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Hospital, Beijing 100080, China
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*Correspondence to: Xiang-Lan Piao, Institute of Chinese Minority Traditional Medicine,
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Minzu University of China. No.27, Zhongguancun South Street, Haidian District, Beijing
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100081, China. E-mail: [email protected], Tel/Fax: +86-10-68939905
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Abstract Folk drug Gynostemma pentaphyllum (Thunb.) Makino contains many biologically active phytochemicals which have been demonstrated to be effective against chronic diseases.
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As in vivo anti-tumor experiments of Gynostemma pentaphyllum extract (GP) show much stronger antitumor activities than in vitro, it is important and necessary to understand the
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metabolic study of GP. A sensitive and specific U-HPLC-MS method was utilized for the
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first time to rapidly identify gypenosides and its possible metabolites in rat serum, urine, and faeces after oral administration. Solid phase extraction was utilized in the sample
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preparation. Negative Electrospray ionisation (ESI) mass spectrometry was used to discern gypenosides and its possible metabolites in rat samples. As a result, after oral
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administration, a total of seven metabolites of G. pentaphyllum extract were assigned,
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two from the rat serum and seven both from the rat urine and faeces. As metabolites of G.
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pentaphyllum extract, all of them have never been reported before. Keywords: Gynostemma pentaphyllum extract (GP); Metabolic profiling; U-HPLC-MS;
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Oral administration
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1
Introduction
The use of ethnobotanical drugs as complementary medicine is provalent in Asia and is also gaining increasing popularity in the west. As one of the well-known traditional
family,
contains
many
biologically
active
phytochemicals
[1].
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Cucurbitaceae
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Chinese herbal medicines, Gynostemma pentaphyllum (Thunb.) Makino, a member of the
Gypenosides, a group of dammarane-type triterpene saponins, are known to be the
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principal bioactive constituents of G. pentaphyllum [2]. As the gypenosides were reported structurally similar to ginsenosides from the expensive ginseng root, G. pentaphyllum
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has attracted much interest as a potential new plant drug. Pharmacological studies of
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gypenosides have shown a variety of interesting biological activities, such as anti-hyperlipidemic [3], hypoglycemic [4], anti-inflammatory [5], and anti-tumor
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activities [6,7]. But previous studies found that in vivo anti-tumor experiments of G.
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pentaphyllum extract (GP) showed much stronger antitumor activities than in vitro. These might suggest that in vivo metabolites of GP played a greater role in anti-tumor activity
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than GP [8-10]. Consequently, in order to understand the pharmacological effects of G. pentaphyllum and explore the real effective components in G. pentaphyllum, it is important and necessary to understand the metabolic study of GP. The metabolism of ginseng and ginsenosides has been investigated extensively in recent years [11-13]. However, to the best of our knowledge, unlike ginsenosides, no data available described the metabolism and metabolites of gypenosides at present. Obviously, the study on metabolism of gypenosides will obtain valuable data and results to facilitate us to better understand the pharmacological or toxicological activities of gypenosides and play a crucial role in the development and clinical application of this potential folk drug.
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According to previous metabolic studies on ginsenosides, the triterpene skeletons of both panaxadiol and panaxatriol were not changed at the process of metabolism [14-16]. Because of the similar structure with ginsenosides, the gypenosides may show the same
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process of metabolism. That is to say, neither parent compounds nor their possible metabolites should have any chromophore in chemical structures. As a result,
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gypenosides and their metabolites mainly show terminal absorptions in their UV spectra.
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Apparently UV detector is not really an optimal tool to detect gypenosides and their metabolites. Among the currently available analytical techniques, many analytical
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methods, like high performance liquid chromatography with ultraviolet detection (HPLC-UV), fluorescence detection (HPLC-FLD), evaporative light-scattering detection
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(HPLC-ELSD), have been reported for the detection of saponins [17-19]. However, most
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of these methods have such disadvantages as long run time, low sensitivity, low
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resolution and high detection limit, failing to meet the requirement of speedy, accurate and high throughput analysis of samples in laboratories. Most of all, they all can hardly
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obtain necessary information about chemical structures of analytes, which causes difficulty in identifying possible metabolites of parent compound. Ultra performance liquid chromatography coupled with tandem mass spectrometry (U-HPLC-MS) utilizes small silica particle column ranging 1.7 µm, which makes possible toper form efficient separations in a short analysis time [13, 20-21]. U-HPLC-MS is a very powerful and reliable tool for the characterization of chemical constituents. And due to its high resolution, sensitivity and specificity, U-HPLC-MS is also suitable for identifying the minor metabolites and has been frequently used in recent metabolism studies of drugs.
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In the present study, a sensitive and specific U-HPLC-MS method was developed for the first time to rapidly identify gypenosides and its possible metabolites in rat serum, urine and faeces. After oral administration, a total of seven metabolites of G. pentaphyllum
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extract were plausibly assigned, two from the rat serum and seven from the rat urine and faeces (Fig. 1). As metabolites of G. pentaphyllum extract, all of them have never been
2.1
Experimental
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reported before.
Chemicals and reagents
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Standards of gypenoside LVI, gypenoside XLVI, gypenoside L, gypenoside LI, damulin
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A, damulin B and 2α-OH-PPD with the purity of ≥ 98.0% were isolated, identified and determined from the raw, heat processed and alkaline hydrolyzed G. pentaphyllum by us
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[6, 22]. Their purities were ≥ 98.0% determined by HPLC-UV analysis. HPLC-grade
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acetonitrile and methanol were all purchased from Fisher Chemical, USA. Water for HPLC analysis was purified using Milli-Q water product (Millipore Corporate, France).
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Ammonium acetate was purchased from Fluka (Burchs, Switzerland). All other reagents were of analytical reagent grade. The leaves of G. pentaphyllum were obtained from Tong Ren Tang (Beijing, China; collected from Fujian province, September). Voucher specimen (No. GP2011-01) was deposited at the Isolation and Structure Identification Laboratory in Minzu University of China, China. 2.2
Preparation of G. pentaphyllum extract (GP)
After extraction in 80% ethanol under reflux condition for 3 h three times, extract was filtered and evaporated. Finally, the extract solution was lyophilized to obtain a powder state of GP. Before orally administrated to the rats, the concentrations of key gypenosides
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in GP were quantified by us. The linear regression, LOQs and LODs, intra-day and inter day precisions and recovery for key gypenosides in extract were shown in Table 1. The concentrations of gypenoside LVI, XLVI and LI were 192.21, 178.13, and 15.26 µg/mg,
Administration
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2.3
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respectively. Gypenoside L, damulin A and damulin B were not detected in GP.
In this study, the experimentation on rats obtained approval from an independent ethics
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committee at Beijing Normal University and the animals received humane care in compliance with international guidelines. Nine healthy male Sprague–Dawley rats (SPF
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degree, body weight 240-260 g) were purchased from Experimental Animal Center of Academy of Military Medical Sciences. Rats were housed in a temperature (23 ± 3 oC)
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and moisture (55 ± 10%) controlled room, exposed to a controlled 12 h cycle of light and
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darkness, and allowed free access to food and water. The rats were halved into three
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groups at random in metabolism cages and fasted for 24 h before use and for 8 h after dosing. The first group was a blank control, whereas the rest two groups were administrated with GP. GP (200 mg/kg), ultra-sonication dissolved with 3 ml water, was orally administrated to the rats. Serum sample was obtained from the second group two hours after administration. In group three, urine and faeces samples were collected from 0-48 h after administration. All samples were stored at -20 oC prior to analysis. 2.4
Sample preparation
After placed at 4 oC for 4 h, the rat blood samples were centrifuged (3,000 rpm) for 10 min, obtaining serum samples (3.5, 3.8, 4.2 ml). Three multiple volume methanol was added to all serum samples which were then vortexed for 2 min before each sample was
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centrifuged (12,000 rpm) for 10 min. Next, three multiple volume methanol was added to above samples and the samples were centrifuged (15,000 rpm) for 10 min after vortexed for 2 min. The supernatant was dried in water-bath (50 oC) and the residue was then
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reconstituted with 1.5 ml methanol. After 20 min treatment with 40 kHz and 500W ultrasonic treatment, the methanol solution was centrifuged (15,000 rpm) for 10 min and
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then the supernatant was microfiltered through 0.22 μm micropore membrane prior to be
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taken for U-HPLC-MS analysis.
Solid-phase extraction (SPE) is an increasingly useful sample preparation technique [23].
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Specific advantages of SPE include easy operation, faster sample processing, good
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purification of compounds from complex samples and procedural simplicity potentially reducing the risk of manipulation errors in routine analysis. Sample preparation of urine
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and faeces was performed by solid phase extraction (SPE) with Cleanert SPE-C18
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cartridges (100 mg/1 ml, Agela Technologies, China). The cartridge was a reverse phase C-18 extraction column used silica gel as the substrate. Its particle size and pore size was
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40-60 µm and 70 Å, respectively.
Each urine sample (25.3, 20.5, 30.6 ml)was concentrated to 10 ml under reduced pressure, and then the concentrated samples (1 ml) were loaded onto Cleanert SPE-C18 cartridges which had been preconditioned with 1 ml methanol and then 1 ml ultrapure water. Each cartridge was washed with 1 ml ultrapure water remove the aqueous effluent and then eluted with 1.5 ml methanol. The eluate was dried in water-bath (50 oC) and the residue was then reconstituted with 1 ml of methanol. Each analyte was microfiltered through 0.22 μm micropore membrane into autosampler vial and 10 µl of the sample was injected into the U-HPLC-MS system for analysis.
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Each faeces sample was refluxed with 80% ethanol for 3 h three times. The solution was concentrated to 10 ml under reduced pressure, and then the concentrated sample (1 ml) was loaded onto a pretreated SPE cartridge. After sample loading, the cartridge was
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washed with 1 ml ultrapure water and eluted with 1.5 ml methanol. The eluate was dried in water-bath (50 oC) and the residue was then reconstituted with 1 ml of methanol. Each
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analyte was microfiltered through 0.22 μm micropore membrane into autosampler vial
Method validation
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2.5
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and 10 µl of the sample was injected into the U-HPLC-MS system for analysis.
Calibration standards were prepared by spiking 50 µl of working solutions into 450 µl of
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rat blank serum, urine and faeces homogenates. Calibration standards were obtained at concentrations of 20, 50, 100, 200, 500,1000 and 2000 ng/ml for each gypenoside. Each
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standard was prepared in triplicate. The regression data, LOQs (S/N = 10) and LODs
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(S/N = 3) of the components were determined by statistical analysis of the concentrations
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of several runs from the UPLC-MS (Table 2-4). Linear calibration curves with correlation coefficients greater than 0.99 were obtained for all analytes in the concentration ranges 20–2000 ng/ml. For the quantified compounds, the LOQ and LOD were 0.10-0.20 ng and 0.045-0.10 ng by U-HPLC-MS.
Intra- and inter-day precisions were determined by assaying standard solution during a single day and on four different days, respectively. As shown in Table 2-4, the overall intra-day variations were less than 4% for gypenoside LVI, gypenoside XLVI, gypenoside L, gypenoside LI, damulin B and damulin A, respectively. And the overall inter-day variations were less than 5% for gypenoside LVI, gypenoside XLVI, gypenoside L, gypenoside LI, damulin B and damulin A, respectively. These results
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demonstrated that the developed method is reproducible with good precision. The accuracy tests were carried out using a recovery test. Recovery of two tested compounds in rat serum were 99.20% and 99.80%, with an R.S.D. of 3.15% and 2.73% (n = 6), in rat urine were 99.0%,
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respectively (Table 2). Recovery of six tested compounds
98.33%, 100.01%, 97.6%, 97.7% and 98.0%, with an R.S.D. of 1.45%, 2.26%, 2.85%,
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2.01%, 1.97% and 2.15% (n = 6), respectively (Table 3). Recovery of six tested
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compounds in rat faeces were 98.4%, 98.2%, 98.2%, 97.2%, 97.6% and 96.0%, with an R.S.D. of 2.93%, 3.15%, 3.46%, 2.86%, 2.38% and 1.97% (n = 6), respectively (Table 4).
U-HPLC-MS analysis
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2.6
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These values indicated that the method is acceptable.
To identify the gypenoside metabolites, chromatographic separation was performed on a
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Acquity U-HPLC-MS System (Waters, USA) including a Binary Solvent Manager, a
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TUV detector, a Sample Manager. The analytical column was AQUITY U-HPLC BEH
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C18 (100 × 2.1 mm, 1.7 μm). A gradient elution system was used for separation of GP and its possible metabolites. The mobile phase consisted of 10 mM Ammonium acetate as A and acetonitrile as B. The solvent gradient program was used as follows: 32% B to 42% B (0-2 min), 48% B (2.01-8 min) , 100% B (8.01-12 min). The flow rate was 0.25 ml/min and the column temperature was set at 25 oC. The U-HPLC was coupled to an triple-quadruple mass spectrometer(Waters, USA). The mass spectrometer was operated in the negative Electrospray ionisation (ESI) mode with following operation conditions: Capillary voltage, 3.0 kV; ion source temperature, 120 oC; desolvation temperature, 350 o
C; desolvation gas flow rate, 700 L/hr; cone gas flow rate, 50 L/hr. The SIR mode were:
m/z 1093.9, cone (V) =50 for gypenoside LVI; m/z 961.8, cone (V) =60 for gypenoside
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XLVI; m/z 799.6, cone (V) =75 for gypenoside L; m/z 799.6, cone (V) =55 for gypenoside LI; m/z 781.6, cone (V) =55 for damulin A; m/z 781.6, cone (V) =55 for damulin B and m/z 781.6, cone (V) =85 for 2α-OH-PPD. The precursor-product ion pairs
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used in MRM mode were: m/z 1093.9→637.16, 1093.9→475.30 for gypenoside LVI; m/z 961.8→637.16, 961.8→475.28 for gypenoside XLVI; m/z 799.6→637.25, 799.6→475.20
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for gypenoside L; m/z 799.6→637.33, 799.6→475.18 for gypenoside LI; m/z
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781.6→619.34, 781.6→457.25 for damulin A; m/z 781.6→619.36, 781.6→457.10 for damulin B and m/z 475.30→391.11 for 2α-OH-PPD. The data were acquired and
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processed with MassLynx Version 4.1 (Waters, USA). The representative MRM
3
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chromatograms for the gypenosides are shown in Figure 2-4. Results and discussion
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In the present study, serum samples were obtained two hours after administration,
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whereas urine and faeces samples were collected from 0-48 h after administration. GP
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and its metabolites were detected by U-HPLC-MS in the negative ion mode. By comparing the SIR chromatograms of blank and treated rat serum, urine, faeces sample, seven possible metabolites are shown in Figure 5. The serum excretion data for gypenoside metabolites following the oral administration of GP are shown in Figure 2. The major compounds absorbed in serum were gypenosides LVI (M1) and XLVI (M2). As the content of LVI in GP is more abundant than that of XLVI, some moieties of LVI may be deglycosylated into XLVI by gastrointestinal (GI) tract after oral administration of GP. Compared with the serum samples, more kinds of gypenoside metabolites were detected in the urine samples. Except LVI and XLVI, five new gypenoside metabolites (M3-M7)
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were also found. From their SIR chromatograms, M3, M4, M5 showed the same quasi-molecular ion peak at m/z 799. Surprisingly, they also possessed the same [M-H-162]- and [M-H-162-162]- ions at m/z 637 and 475. According to the standard, M4
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was identified as gypenoside L. M5 was plausibly assigned as gypenoside LI, which resulted from isomerization from configuration S to R at C-20 position possibly occurring
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in the process of metabolism with the loss of a glucose moiety [12, 24]. Different from
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gypenoside L and LI, both of which had two glucose residues in C-3 position, M3 should contain two glucose residues but these two glucose residues might be linked to C-3 and
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C-20 respectively, or two glucose residues were both in C-20 position. Consequently, M3, M4 and M5 were further deglycosylated metabolites of gypenoside LVI (M1) and XLVI
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(M2). In addition, M6 and M7 showed the same [M-H]-, [M-H-162]- and [M-H-162-162]-
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ions at m/z 781, 619 and 457. According to the standard, they were identified as damulin
gypenoside L and LI.
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A and B, which resulted from dehydration reaction on the aliphatic side chain of
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Similar to the urinary excretion data, seven same gypenosides metabolites were found in the faeces samples. From their SIR chromatograms, the contents of LVI and XLVI in faeces samples were less than those in urine and serum samples whereas the contents of damulin A and B in faeces samples were richer than those in urine samples. A portion of the excreted gypenosides may not be absorbed from the gastrointestinal tract but directly excreted into the faeces. Regrettably, 2α-OH-Rh2 and 2α-OH-PPD were not detected in all rat samples, which may depend on the low in vivo doses or low oral bioavailability. Since there is no reference available about the in vivo doses of GP, we referred to reported doses used for
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metabolic studies on ginsenosides. Metabolic studies with ginsenosides have been conducted at doses ranging from 50 to 400 mg/kg [12, 25]. Kim and her colleagues used a higher dose than the reported pharmacological doses so as to detect and quantify minor
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metabolites considering that ginsenoside Re shows low intestinal absorption [26]. In their study, ginsenoside Re (pure compound) was orally administered to rats at a dose of 200
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mg/kg. Due to structural similarity to ginsenosides, gypenosides also show low intestinal
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absorption. In this study, G. pentaphyllum extract was orally administered to rats at a dose of 200 mg/kg. Such a low in vivo dose may not be able to detect some minor
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metabolites, such as 2α-OH-Rh2 or 2α-OH-PPD. In order to detect the presence of these
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minor metabolites, further metabolic study must increase the administration dose. Previous studies of the structure–activity relationship between the ginsenosides and
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antitumor activity showed that the aglycones were more effective than the glycosides, and
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that the presence of sugar moieties reduced the antitumor activities [27]. Because of its similar structure with ginsenosides, we predicted that the gypenosides show the similar
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structure–activity relationship. From our reported data[6, 22], we found that damulin B, damulinA, gypenoside L and gypenoside LI, the major metabolites in rat urine and faeces, showed stronger cytotoxic activity against non-small cell lung carcinoma A549 cells than gypenoside LVI and XLVI, the major gypenosides in G. pentaphyllum extract. Such important discoveries may be the reason why in vivo anti-tumor experiments of G. pentaphyllum extract show much stronger antitumor activities than in vitro. This means that when the rats were orally administrated with G. pentaphyllum extract, the major gypenosides LVI and XLVI can be metabolized into damulin B, damulinA, gypenoside L, gypenoside LI and then play a role of anti-tumor activities. Consequently, this study on
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in vivo metabolism of gypenosides will play a valuable role in the development of this potential new drug because it can obtain valuable data and results to facilitate us to better understand the pharmacological or toxicological activities of gypenosides. The proposed
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metabolic pathways of G. pentaphyllum extract in rats are shown in Figure 6.
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However, in this study, the rats were orally administrated with G. pentaphyllum extract, which is a mixture of many different kinds of compositions and is complex to analyze. Its
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possible metabolites were complicated and undefined and we could just identify some deglycosylated productions, a small part of total metabolites. So it is difficult to
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determine the real pathways of the metabolization of parent compounds into metabolites
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and only the proposed metabolic pathways can be speculated Therefore, in order to detect other type metabolites, like oxidation or combinative production, and explore the real
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4. Conclusions
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metabolic pathways, pure compounds should be applied in the future metabolic study.
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In the present study, a rapid, sensitive and specific U-HPLC–ESI–MS method for the identification of gypenosides and its metabolites in rat serum, urine, faeces was developed. Two, seven, and seven metabolites were detected and identified in rat serum, urine and faeces, respectively. Combining with our previous studies [6, 22], the metabolites, damulin B, damulinA, gypenoside L and gypenoside LI, were found to possess stronger cytotoxic activity against A549 cells than parent compounds. The generated gypenosides may be developed to be some kind of promising anti-tumor drug. Consequently, by studying metabolism of GP and identifying its possible metabolites, we can define its real pharmacological effects and lay the foundation for the preparation of anti-tumor drugs with high efficiency and low toxicity.
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flavonoids in the leaves of Isatis indigatica Fort. by ultra-performance liquid chromatography with PDA and electrospray ionization tandem mass spectrometry detection, J. Pharm. Biomed. Anal. 48 (2008) 562–567.
Page 16 of 31
[21]
M. Iqbal, T.A. Wani, N.Y. Khalil, I.A. Darwish, Development and validation of ultra-performance liquid chromatographic method with tandem mass spectrometry for determination of lenalidomide in rabbit and human plasma, Chem. Cent. J. 7
[22]
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(2013) 7.
D.J. Chen, H.M. Liu, S.F. Xing, X.L. Piao, Cytotoxic activity of gypenosides and
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gynogenin against non-small cell lung carcinoma A549 cells, Bioorg. Med. Chem.
[23]
us
Lett. 24 (2014) 186–191.
L. Liu, J. Huang, X Hu, K. Li, C. Sun, Simultaneous determination of ginsenoside
an
(G-Re, G-Rg1, G-Rg2, G-F1, G-Rh1) and protopanaxatriol in human plasma and urine by LC-MS/MS and its application in a pharmacokinetics study of G-Re in
M
volunteers, J. Chromatogr. B. Analyt. Technol. Biomed. Life. Sci. 879 (2011)
H.M. Liu, Y.J. Gao, X.L. Piao, Microbial transformation of gypenoside XLVI by
te
[24]
d
2011–2017.
Lactobacillus delbrueckii subsp. Bulgaricus, Food Science 35 (2014) 45 51. G. Chen, M. Yang, Y. Song, Z. Lu, J. Zhang, H. Huang, S. Guan, L. Wu, D.A. Guo,
Ac ce p
[25]
Comparative analysis on microbial and rat metabolism of ginsenoside Rb1 by
high-performance liquid chromatography coupled with tandem mass spectrometry, Biomed. Chromatogr. 22 (2008) 779–785.
[26]
U. Kim, M.H. Park, D.H. Kim, H.H. Yoo, Metabolite profiling of ginsenoside Re
in rat urine and faeces after oral administration, Food. Chem. 136 (2013) 1364–1369. [27]
D.G. Popovich, D.D. Kitts, Structure-function relationship exists for ginsenosides in reducing cell proliferation and inducing apoptosis in the human leukemia
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te
d
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an
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(THP-1) cell line, Arch. Biochem. Biophys. 406 (2002) 1–8.
Page 18 of 31
Figure captions Figure 1. Structures of possible metabolites of G. pentaphyllum extract Figure 2. Representative MRM chromatograms for the gypenosides in serum samples
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Figure 3. Representative MRM chromatograms for the gypenosides in urine samples
Figure 4. Representative MRM chromatograms for the gypenosides in faeces samples
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Figure 5. SIR chromatograms of blank and treated rat serum, urine, faeces samples
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Figure 6. Proposed metabolic pathways of G. pentaphyllum extract in rats
Page 19 of 31
Highlights ►A sensitive and specific UPLC/MS method was utilized. ►It can rapidly identify metabolites of Gynostemma pentaphyllum extract in rat samples. ►A total of seven
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metabolites of G. Pentaphyllum extract (GP) were assigned.►Two were from the rat serum and seven were both from the rat urine and faeces. ► The proposed metabolic
Ac ce p
te
d
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an
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pathways of GP in rats were explored.
Page 20 of 31
Figure
Ac
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ed
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an
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Figure 1
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ed
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an
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Figure 2
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ed
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an
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Figure 3
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ed
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an
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Figure 4
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an
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Figure 5
Page 25 of 31
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Figure 6
Page 26 of 31
cr
i Compound
Linear regression equation
r
us
Linear regression, LOQs and LODs, intra-day and inter day precisions and recovery for six compounds in extract Linear
LOQ (ng)
LOD (ng)
range
(S/N
(S/N = 3)
(ng/ml)
10)
=
an
Table 1
Intra-day precision RSD (%, n=6)
Inter-day Recovery (n =6) precision Original Added RSD (%, (μg/mg) (μg) n=4)
Deteceted Recovery RSD (μg) (%) (%)
0.09
0.045
1.89
3.52
192.21
200.00
386.34
97.07
2.13
0.10
0.08
3.15
3.44
178.13
200.00
373.78
97.83
1.79
0.12
0. 05
2.71
3.05
0
0.10
0. 045
1.89
3.56
15.26
20.00
35.15
99.45
2.32
20-2000
0.16
0. 08
2.74
3.52
0
20-2000
0.18
0. 09
2.32
2.81
0
Y=0.154236X-0.4043
0.9962
20-2000
gypenoside XLVI
Y=0.147324X-1.1907
0.9992
20-2000
gypenoside L
Y=0.12199X+1.0014
0.9956
20-2000
gypenoside LI
Y=0.103574X-0.0322
0.9960
20-2000
damulin B
Y= 0.117337X+0.0189
0.9972
damulin A
Y=0.0904974X+1.1206
0.9905
Ac
ce
pt
ed
M
gypenoside LVI
Page 27 of 31 1
cr
i Compound
Linear regression equation
r
us
Linear regression, LOQs and LODs, intra-day and inter day precisions and recovery for two compounds in serum Linear
LOQ (ng)
LOD (ng)
range
(S/N
(S/N = 3)
(ng/ml)
10)
Y=0.0163657X-0.6543
0.9977
20-2000
gypenoside XLVI
Y=0.0156428X-1.4352
0.9985
20-2000
Intra-day precision RSD (%, n=6)
Inter-day Recovery (n =6) precision Original Added RSD (%, (μg) (μg) n=4)
Deteceted Recovery RSD (μg) (%) (%)
0.18
0.07
1.93
3.86
6.09
10.00
16.01
99.2
3.15
0.20
0.08
2.85
4.13
10.13
10.00
20.01.
98.8
2.73
Ac
ce
pt
ed
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gypenoside LVI
=
an
Table 2
Page 28 of 31 2
cr
i Compound
Linear regression equation
r
us
Linear regression, LOQs and LODs, intra-day and inter day precisions and recovery for six compounds in urine Linear
LOQ (ng)
LOD (ng)
range
(S/N
(S/N = 3)
(ng/ml)
10)
=
an
Table 3
Intra-day precision RSD (%, n=6)
Inter-day Recovery (n =6) precision Original Added RSD (%, (μg) (μg) n=4)
Deteceted Recovery RSD (μg) (%) (%)
0.16
0.065
3.92
3.40
1.69
2.00
3.61
96.0
1.45
0.20
0.08
3.50
3.74
2 .70
3.00
5.65
98.33
2.26
0.14
0. 05
3.77
2.57
2.65
2.00
4.73
100.01
2.85
20-2000
0.10
0. 045
2.55
4.37
3.17
3.00
6.07
97.6
2.01
0.9989
20-2000
0.20
0. 08
2.75
4.51
2.81
3.00
5.74
97.7
1.97
0.9960
20-2000
0.19
0. 09
3.73
4.61
1.65
2.00
3.61
98.0
2.15
Y=0.0168845X+0.3293
0.9969
20-2000
gypenoside XLVI
Y=0.0147866X-1.8904
0.9986
20-2000
gypenoside L
Y=0.0672750X+0.5929
0.9913
20-2000
gypenoside LI
Y=0.0982409X-0.1259
0.9928
damulin B
Y= 0.0708798X-0.0930
damulin A
Y=0.0252250X+0.3946
Ac
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pt
ed
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gypenoside LVI
Page 29 of 31 3
cr
i Compound
Linear regression equation
r
us
Linear regression, LOQs and LODs, intra-day and inter day precisions and recovery for six compounds in faeces Linear
LOQ (ng)
LOD (ng)
range
(S/N
(S/N = 3)
(ng/ml)
10)
=
an
Table 4
Intra-day precision RSD (%, n=6)
Inter-day Recovery (n =6 precision Original Added RSD (%, (μg) (μg) n=4)
Deteceted Recovery RSD (μg) (%) (%)
0.20
0.08
3.72
4.29
4.43
5.00
9.35
98.4
2.93
0.15
0.09
3.82
4.09
4.95
5.00
9.86
98.2
3.15
0.16
0. 055
2.65
4.56
3.58
5.00
8.49
98.2
3.46
20-2000
0.15
0. 065
2.84
3.66
6.33
5.00
11.19
97.2
2.86
0.9981
20-2000
0.18
0. 09
2.39
3.18
5.86
5.00
10.74
97.6
2.38
0.9981
20-2000
0.20
0. 10
2.21
2.66
1.24
2.00
3.16
96.0
1.97
Y=0.0163226X+0.9140
0.9976
20-2000
gypenoside XLVI
Y=0.0166629X-1.2393
0.9965
20-2000
gypenoside L
Y=0.0902131X-0.1186
0.9917
20-2000
gypenoside LI
Y=0.0931877X-0.1126
0.9988
damulin B
Y= 0.0664668X+0.1579
damulin A
Y=0.1056950X-0.2134
Ac
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gypenoside LVI
Page 30 of 31 4
i cr
Metabolites in the urine samples (ng/ml) 1
c
2
3
Mean
gypenoside LVI
67.10
94.55
23.23
61.63
gypenoside XLVI
117.40
131.79
65.97
195.05
gypenoside L
127.41
185.36
86.73
133.17
gypenoside LI
94.58
154.68
66.46
105.24
damulin B
111.00
104.25
66.25
93.83
damulin A
65.13
72.71
46.28
61.37
Metabolites in the faeces samples (ng/mg) 1
d
2
an
Gypenosides
us
Excretion of gypenoside metabolites in the serum, urine and faeces after the oral administration of GP.a
Each rat was administered on average 50 mg of GP.
b
Not detected, which is considered as 0.
c
Volume of urine sample (25.3, 20.5, 30.6 ml)
d
Weight of faeces sample (8530.2, 9380.4, 9873.5 mg)
e
Volume of serum sample (3.5, 3.8, 4.2 ml)
3
Mean
1
e
2
3
Mean
0.44
0.38
0.45
1740.01
1522.86
1615.55
1626.14
0.57
0.52
0.50
0.53
2894.36
2593.77
2696.94
2728.35
b
0.42
0.80
0.46
0.56
ND
ND
ND
ND
0.61
1.14
0.64
0.80
ND
ND
ND
ND
0.56
0.84
0.59
0.67
ND
ND
ND
ND
0.14
0.14
0.07
0.12
ND
ND
ND
ND
Ac
ce
pt
ed
a
Metabolites in the serum samples (ng/ml)
0.52
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Table 5
Page 31 of 31 5
S1570-0232(14)00512-1 http://dx.doi.org/doi:10.1016/j.jchromb.2014.08.003 CHROMB 19065
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
17-3-2014 31-7-2014 3-8-2014
Please cite this article as: D.-J. Chen, H.-G. Hu, S.-F. Xing, Y.-J. Gao, S.-F. Xu, X.-L. Piao, Metabolic profiling of Gynostemma pentaphyllum extract in rat serum, urine and faeces after oral administration, Journal of Chromatography B (2014), http://dx.doi.org/10.1016/j.jchromb.2014.08.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Metabolic profiling of Gynostemma pentaphyllum extract in rat serum, urine and faeces after oral administration
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Dao-Jin Chena, Hua-Gang Hua, Shao-Fang Xinga, Ya-Jun Gaob, Si-Fan Xua, Xiang-Lan
Institute of Chinese Minority Traditional Medicine, Minzu University of China, Beijing
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a
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Piaoa,*
100081, China
Department of Breast Disease Prevention Center, Haidian Maternal and Child Health
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b
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Hospital, Beijing 100080, China
d
*Correspondence to: Xiang-Lan Piao, Institute of Chinese Minority Traditional Medicine,
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Minzu University of China. No.27, Zhongguancun South Street, Haidian District, Beijing
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100081, China. E-mail: [email protected], Tel/Fax: +86-10-68939905
Page 1 of 31
Abstract Folk drug Gynostemma pentaphyllum (Thunb.) Makino contains many biologically active phytochemicals which have been demonstrated to be effective against chronic diseases.
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As in vivo anti-tumor experiments of Gynostemma pentaphyllum extract (GP) show much stronger antitumor activities than in vitro, it is important and necessary to understand the
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metabolic study of GP. A sensitive and specific U-HPLC-MS method was utilized for the
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first time to rapidly identify gypenosides and its possible metabolites in rat serum, urine, and faeces after oral administration. Solid phase extraction was utilized in the sample
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preparation. Negative Electrospray ionisation (ESI) mass spectrometry was used to discern gypenosides and its possible metabolites in rat samples. As a result, after oral
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administration, a total of seven metabolites of G. pentaphyllum extract were assigned,
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two from the rat serum and seven both from the rat urine and faeces. As metabolites of G.
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pentaphyllum extract, all of them have never been reported before. Keywords: Gynostemma pentaphyllum extract (GP); Metabolic profiling; U-HPLC-MS;
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Oral administration
Page 2 of 31
1
Introduction
The use of ethnobotanical drugs as complementary medicine is provalent in Asia and is also gaining increasing popularity in the west. As one of the well-known traditional
family,
contains
many
biologically
active
phytochemicals
[1].
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Cucurbitaceae
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Chinese herbal medicines, Gynostemma pentaphyllum (Thunb.) Makino, a member of the
Gypenosides, a group of dammarane-type triterpene saponins, are known to be the
us
principal bioactive constituents of G. pentaphyllum [2]. As the gypenosides were reported structurally similar to ginsenosides from the expensive ginseng root, G. pentaphyllum
an
has attracted much interest as a potential new plant drug. Pharmacological studies of
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gypenosides have shown a variety of interesting biological activities, such as anti-hyperlipidemic [3], hypoglycemic [4], anti-inflammatory [5], and anti-tumor
d
activities [6,7]. But previous studies found that in vivo anti-tumor experiments of G.
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pentaphyllum extract (GP) showed much stronger antitumor activities than in vitro. These might suggest that in vivo metabolites of GP played a greater role in anti-tumor activity
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than GP [8-10]. Consequently, in order to understand the pharmacological effects of G. pentaphyllum and explore the real effective components in G. pentaphyllum, it is important and necessary to understand the metabolic study of GP. The metabolism of ginseng and ginsenosides has been investigated extensively in recent years [11-13]. However, to the best of our knowledge, unlike ginsenosides, no data available described the metabolism and metabolites of gypenosides at present. Obviously, the study on metabolism of gypenosides will obtain valuable data and results to facilitate us to better understand the pharmacological or toxicological activities of gypenosides and play a crucial role in the development and clinical application of this potential folk drug.
Page 3 of 31
According to previous metabolic studies on ginsenosides, the triterpene skeletons of both panaxadiol and panaxatriol were not changed at the process of metabolism [14-16]. Because of the similar structure with ginsenosides, the gypenosides may show the same
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process of metabolism. That is to say, neither parent compounds nor their possible metabolites should have any chromophore in chemical structures. As a result,
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gypenosides and their metabolites mainly show terminal absorptions in their UV spectra.
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Apparently UV detector is not really an optimal tool to detect gypenosides and their metabolites. Among the currently available analytical techniques, many analytical
an
methods, like high performance liquid chromatography with ultraviolet detection (HPLC-UV), fluorescence detection (HPLC-FLD), evaporative light-scattering detection
M
(HPLC-ELSD), have been reported for the detection of saponins [17-19]. However, most
d
of these methods have such disadvantages as long run time, low sensitivity, low
te
resolution and high detection limit, failing to meet the requirement of speedy, accurate and high throughput analysis of samples in laboratories. Most of all, they all can hardly
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obtain necessary information about chemical structures of analytes, which causes difficulty in identifying possible metabolites of parent compound. Ultra performance liquid chromatography coupled with tandem mass spectrometry (U-HPLC-MS) utilizes small silica particle column ranging 1.7 µm, which makes possible toper form efficient separations in a short analysis time [13, 20-21]. U-HPLC-MS is a very powerful and reliable tool for the characterization of chemical constituents. And due to its high resolution, sensitivity and specificity, U-HPLC-MS is also suitable for identifying the minor metabolites and has been frequently used in recent metabolism studies of drugs.
Page 4 of 31
In the present study, a sensitive and specific U-HPLC-MS method was developed for the first time to rapidly identify gypenosides and its possible metabolites in rat serum, urine and faeces. After oral administration, a total of seven metabolites of G. pentaphyllum
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extract were plausibly assigned, two from the rat serum and seven from the rat urine and faeces (Fig. 1). As metabolites of G. pentaphyllum extract, all of them have never been
2.1
Experimental
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2
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reported before.
Chemicals and reagents
an
Standards of gypenoside LVI, gypenoside XLVI, gypenoside L, gypenoside LI, damulin
M
A, damulin B and 2α-OH-PPD with the purity of ≥ 98.0% were isolated, identified and determined from the raw, heat processed and alkaline hydrolyzed G. pentaphyllum by us
d
[6, 22]. Their purities were ≥ 98.0% determined by HPLC-UV analysis. HPLC-grade
te
acetonitrile and methanol were all purchased from Fisher Chemical, USA. Water for HPLC analysis was purified using Milli-Q water product (Millipore Corporate, France).
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Ammonium acetate was purchased from Fluka (Burchs, Switzerland). All other reagents were of analytical reagent grade. The leaves of G. pentaphyllum were obtained from Tong Ren Tang (Beijing, China; collected from Fujian province, September). Voucher specimen (No. GP2011-01) was deposited at the Isolation and Structure Identification Laboratory in Minzu University of China, China. 2.2
Preparation of G. pentaphyllum extract (GP)
After extraction in 80% ethanol under reflux condition for 3 h three times, extract was filtered and evaporated. Finally, the extract solution was lyophilized to obtain a powder state of GP. Before orally administrated to the rats, the concentrations of key gypenosides
Page 5 of 31
in GP were quantified by us. The linear regression, LOQs and LODs, intra-day and inter day precisions and recovery for key gypenosides in extract were shown in Table 1. The concentrations of gypenoside LVI, XLVI and LI were 192.21, 178.13, and 15.26 µg/mg,
Administration
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2.3
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respectively. Gypenoside L, damulin A and damulin B were not detected in GP.
In this study, the experimentation on rats obtained approval from an independent ethics
an
committee at Beijing Normal University and the animals received humane care in compliance with international guidelines. Nine healthy male Sprague–Dawley rats (SPF
M
degree, body weight 240-260 g) were purchased from Experimental Animal Center of Academy of Military Medical Sciences. Rats were housed in a temperature (23 ± 3 oC)
d
and moisture (55 ± 10%) controlled room, exposed to a controlled 12 h cycle of light and
te
darkness, and allowed free access to food and water. The rats were halved into three
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groups at random in metabolism cages and fasted for 24 h before use and for 8 h after dosing. The first group was a blank control, whereas the rest two groups were administrated with GP. GP (200 mg/kg), ultra-sonication dissolved with 3 ml water, was orally administrated to the rats. Serum sample was obtained from the second group two hours after administration. In group three, urine and faeces samples were collected from 0-48 h after administration. All samples were stored at -20 oC prior to analysis. 2.4
Sample preparation
After placed at 4 oC for 4 h, the rat blood samples were centrifuged (3,000 rpm) for 10 min, obtaining serum samples (3.5, 3.8, 4.2 ml). Three multiple volume methanol was added to all serum samples which were then vortexed for 2 min before each sample was
Page 6 of 31
centrifuged (12,000 rpm) for 10 min. Next, three multiple volume methanol was added to above samples and the samples were centrifuged (15,000 rpm) for 10 min after vortexed for 2 min. The supernatant was dried in water-bath (50 oC) and the residue was then
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reconstituted with 1.5 ml methanol. After 20 min treatment with 40 kHz and 500W ultrasonic treatment, the methanol solution was centrifuged (15,000 rpm) for 10 min and
cr
then the supernatant was microfiltered through 0.22 μm micropore membrane prior to be
us
taken for U-HPLC-MS analysis.
Solid-phase extraction (SPE) is an increasingly useful sample preparation technique [23].
an
Specific advantages of SPE include easy operation, faster sample processing, good
M
purification of compounds from complex samples and procedural simplicity potentially reducing the risk of manipulation errors in routine analysis. Sample preparation of urine
d
and faeces was performed by solid phase extraction (SPE) with Cleanert SPE-C18
te
cartridges (100 mg/1 ml, Agela Technologies, China). The cartridge was a reverse phase C-18 extraction column used silica gel as the substrate. Its particle size and pore size was
Ac ce p
40-60 µm and 70 Å, respectively.
Each urine sample (25.3, 20.5, 30.6 ml)was concentrated to 10 ml under reduced pressure, and then the concentrated samples (1 ml) were loaded onto Cleanert SPE-C18 cartridges which had been preconditioned with 1 ml methanol and then 1 ml ultrapure water. Each cartridge was washed with 1 ml ultrapure water remove the aqueous effluent and then eluted with 1.5 ml methanol. The eluate was dried in water-bath (50 oC) and the residue was then reconstituted with 1 ml of methanol. Each analyte was microfiltered through 0.22 μm micropore membrane into autosampler vial and 10 µl of the sample was injected into the U-HPLC-MS system for analysis.
Page 7 of 31
Each faeces sample was refluxed with 80% ethanol for 3 h three times. The solution was concentrated to 10 ml under reduced pressure, and then the concentrated sample (1 ml) was loaded onto a pretreated SPE cartridge. After sample loading, the cartridge was
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washed with 1 ml ultrapure water and eluted with 1.5 ml methanol. The eluate was dried in water-bath (50 oC) and the residue was then reconstituted with 1 ml of methanol. Each
cr
analyte was microfiltered through 0.22 μm micropore membrane into autosampler vial
Method validation
an
2.5
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and 10 µl of the sample was injected into the U-HPLC-MS system for analysis.
Calibration standards were prepared by spiking 50 µl of working solutions into 450 µl of
M
rat blank serum, urine and faeces homogenates. Calibration standards were obtained at concentrations of 20, 50, 100, 200, 500,1000 and 2000 ng/ml for each gypenoside. Each
d
standard was prepared in triplicate. The regression data, LOQs (S/N = 10) and LODs
te
(S/N = 3) of the components were determined by statistical analysis of the concentrations
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of several runs from the UPLC-MS (Table 2-4). Linear calibration curves with correlation coefficients greater than 0.99 were obtained for all analytes in the concentration ranges 20–2000 ng/ml. For the quantified compounds, the LOQ and LOD were 0.10-0.20 ng and 0.045-0.10 ng by U-HPLC-MS.
Intra- and inter-day precisions were determined by assaying standard solution during a single day and on four different days, respectively. As shown in Table 2-4, the overall intra-day variations were less than 4% for gypenoside LVI, gypenoside XLVI, gypenoside L, gypenoside LI, damulin B and damulin A, respectively. And the overall inter-day variations were less than 5% for gypenoside LVI, gypenoside XLVI, gypenoside L, gypenoside LI, damulin B and damulin A, respectively. These results
Page 8 of 31
demonstrated that the developed method is reproducible with good precision. The accuracy tests were carried out using a recovery test. Recovery of two tested compounds in rat serum were 99.20% and 99.80%, with an R.S.D. of 3.15% and 2.73% (n = 6), in rat urine were 99.0%,
ip t
respectively (Table 2). Recovery of six tested compounds
98.33%, 100.01%, 97.6%, 97.7% and 98.0%, with an R.S.D. of 1.45%, 2.26%, 2.85%,
cr
2.01%, 1.97% and 2.15% (n = 6), respectively (Table 3). Recovery of six tested
us
compounds in rat faeces were 98.4%, 98.2%, 98.2%, 97.2%, 97.6% and 96.0%, with an R.S.D. of 2.93%, 3.15%, 3.46%, 2.86%, 2.38% and 1.97% (n = 6), respectively (Table 4).
U-HPLC-MS analysis
M
2.6
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These values indicated that the method is acceptable.
To identify the gypenoside metabolites, chromatographic separation was performed on a
d
Acquity U-HPLC-MS System (Waters, USA) including a Binary Solvent Manager, a
te
TUV detector, a Sample Manager. The analytical column was AQUITY U-HPLC BEH
Ac ce p
C18 (100 × 2.1 mm, 1.7 μm). A gradient elution system was used for separation of GP and its possible metabolites. The mobile phase consisted of 10 mM Ammonium acetate as A and acetonitrile as B. The solvent gradient program was used as follows: 32% B to 42% B (0-2 min), 48% B (2.01-8 min) , 100% B (8.01-12 min). The flow rate was 0.25 ml/min and the column temperature was set at 25 oC. The U-HPLC was coupled to an triple-quadruple mass spectrometer(Waters, USA). The mass spectrometer was operated in the negative Electrospray ionisation (ESI) mode with following operation conditions: Capillary voltage, 3.0 kV; ion source temperature, 120 oC; desolvation temperature, 350 o
C; desolvation gas flow rate, 700 L/hr; cone gas flow rate, 50 L/hr. The SIR mode were:
m/z 1093.9, cone (V) =50 for gypenoside LVI; m/z 961.8, cone (V) =60 for gypenoside
Page 9 of 31
XLVI; m/z 799.6, cone (V) =75 for gypenoside L; m/z 799.6, cone (V) =55 for gypenoside LI; m/z 781.6, cone (V) =55 for damulin A; m/z 781.6, cone (V) =55 for damulin B and m/z 781.6, cone (V) =85 for 2α-OH-PPD. The precursor-product ion pairs
ip t
used in MRM mode were: m/z 1093.9→637.16, 1093.9→475.30 for gypenoside LVI; m/z 961.8→637.16, 961.8→475.28 for gypenoside XLVI; m/z 799.6→637.25, 799.6→475.20
cr
for gypenoside L; m/z 799.6→637.33, 799.6→475.18 for gypenoside LI; m/z
us
781.6→619.34, 781.6→457.25 for damulin A; m/z 781.6→619.36, 781.6→457.10 for damulin B and m/z 475.30→391.11 for 2α-OH-PPD. The data were acquired and
an
processed with MassLynx Version 4.1 (Waters, USA). The representative MRM
3
M
chromatograms for the gypenosides are shown in Figure 2-4. Results and discussion
d
In the present study, serum samples were obtained two hours after administration,
te
whereas urine and faeces samples were collected from 0-48 h after administration. GP
Ac ce p
and its metabolites were detected by U-HPLC-MS in the negative ion mode. By comparing the SIR chromatograms of blank and treated rat serum, urine, faeces sample, seven possible metabolites are shown in Figure 5. The serum excretion data for gypenoside metabolites following the oral administration of GP are shown in Figure 2. The major compounds absorbed in serum were gypenosides LVI (M1) and XLVI (M2). As the content of LVI in GP is more abundant than that of XLVI, some moieties of LVI may be deglycosylated into XLVI by gastrointestinal (GI) tract after oral administration of GP. Compared with the serum samples, more kinds of gypenoside metabolites were detected in the urine samples. Except LVI and XLVI, five new gypenoside metabolites (M3-M7)
Page 10 of 31
were also found. From their SIR chromatograms, M3, M4, M5 showed the same quasi-molecular ion peak at m/z 799. Surprisingly, they also possessed the same [M-H-162]- and [M-H-162-162]- ions at m/z 637 and 475. According to the standard, M4
ip t
was identified as gypenoside L. M5 was plausibly assigned as gypenoside LI, which resulted from isomerization from configuration S to R at C-20 position possibly occurring
cr
in the process of metabolism with the loss of a glucose moiety [12, 24]. Different from
us
gypenoside L and LI, both of which had two glucose residues in C-3 position, M3 should contain two glucose residues but these two glucose residues might be linked to C-3 and
an
C-20 respectively, or two glucose residues were both in C-20 position. Consequently, M3, M4 and M5 were further deglycosylated metabolites of gypenoside LVI (M1) and XLVI
M
(M2). In addition, M6 and M7 showed the same [M-H]-, [M-H-162]- and [M-H-162-162]-
d
ions at m/z 781, 619 and 457. According to the standard, they were identified as damulin
gypenoside L and LI.
te
A and B, which resulted from dehydration reaction on the aliphatic side chain of
Ac ce p
Similar to the urinary excretion data, seven same gypenosides metabolites were found in the faeces samples. From their SIR chromatograms, the contents of LVI and XLVI in faeces samples were less than those in urine and serum samples whereas the contents of damulin A and B in faeces samples were richer than those in urine samples. A portion of the excreted gypenosides may not be absorbed from the gastrointestinal tract but directly excreted into the faeces. Regrettably, 2α-OH-Rh2 and 2α-OH-PPD were not detected in all rat samples, which may depend on the low in vivo doses or low oral bioavailability. Since there is no reference available about the in vivo doses of GP, we referred to reported doses used for
Page 11 of 31
metabolic studies on ginsenosides. Metabolic studies with ginsenosides have been conducted at doses ranging from 50 to 400 mg/kg [12, 25]. Kim and her colleagues used a higher dose than the reported pharmacological doses so as to detect and quantify minor
ip t
metabolites considering that ginsenoside Re shows low intestinal absorption [26]. In their study, ginsenoside Re (pure compound) was orally administered to rats at a dose of 200
cr
mg/kg. Due to structural similarity to ginsenosides, gypenosides also show low intestinal
us
absorption. In this study, G. pentaphyllum extract was orally administered to rats at a dose of 200 mg/kg. Such a low in vivo dose may not be able to detect some minor
an
metabolites, such as 2α-OH-Rh2 or 2α-OH-PPD. In order to detect the presence of these
M
minor metabolites, further metabolic study must increase the administration dose. Previous studies of the structure–activity relationship between the ginsenosides and
d
antitumor activity showed that the aglycones were more effective than the glycosides, and
te
that the presence of sugar moieties reduced the antitumor activities [27]. Because of its similar structure with ginsenosides, we predicted that the gypenosides show the similar
Ac ce p
structure–activity relationship. From our reported data[6, 22], we found that damulin B, damulinA, gypenoside L and gypenoside LI, the major metabolites in rat urine and faeces, showed stronger cytotoxic activity against non-small cell lung carcinoma A549 cells than gypenoside LVI and XLVI, the major gypenosides in G. pentaphyllum extract. Such important discoveries may be the reason why in vivo anti-tumor experiments of G. pentaphyllum extract show much stronger antitumor activities than in vitro. This means that when the rats were orally administrated with G. pentaphyllum extract, the major gypenosides LVI and XLVI can be metabolized into damulin B, damulinA, gypenoside L, gypenoside LI and then play a role of anti-tumor activities. Consequently, this study on
Page 12 of 31
in vivo metabolism of gypenosides will play a valuable role in the development of this potential new drug because it can obtain valuable data and results to facilitate us to better understand the pharmacological or toxicological activities of gypenosides. The proposed
ip t
metabolic pathways of G. pentaphyllum extract in rats are shown in Figure 6.
cr
However, in this study, the rats were orally administrated with G. pentaphyllum extract, which is a mixture of many different kinds of compositions and is complex to analyze. Its
us
possible metabolites were complicated and undefined and we could just identify some deglycosylated productions, a small part of total metabolites. So it is difficult to
an
determine the real pathways of the metabolization of parent compounds into metabolites
M
and only the proposed metabolic pathways can be speculated Therefore, in order to detect other type metabolites, like oxidation or combinative production, and explore the real
te
4. Conclusions
d
metabolic pathways, pure compounds should be applied in the future metabolic study.
Ac ce p
In the present study, a rapid, sensitive and specific U-HPLC–ESI–MS method for the identification of gypenosides and its metabolites in rat serum, urine, faeces was developed. Two, seven, and seven metabolites were detected and identified in rat serum, urine and faeces, respectively. Combining with our previous studies [6, 22], the metabolites, damulin B, damulinA, gypenoside L and gypenoside LI, were found to possess stronger cytotoxic activity against A549 cells than parent compounds. The generated gypenosides may be developed to be some kind of promising anti-tumor drug. Consequently, by studying metabolism of GP and identifying its possible metabolites, we can define its real pharmacological effects and lay the foundation for the preparation of anti-tumor drugs with high efficiency and low toxicity.
Page 13 of 31
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Lee, K.M. Lim, 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. 51 (2010) 278–283.
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L. Yang, S.J. Xu , X. Zeng, Z.F. Wu, Y.H. Deng, Y.M. Liu, S.G. Deng, R.M. Ou, In vivo rat metabolism studies of ginsenoside Rb1, Chem. J. Chin. Univ. 27 (2006) 1042–1044. E.A. Bae, M.J Han, M.K. Choo, S.Y. Park, D.H. Kim, Metabolism of 20(S)- and
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20(R)-ginsenoside Rg3 by human intestinal bacteria and its relation to in vitro
L. Yang, Y. Deng, S Xu, X. Zeng, In vivo pharmacokinetic and metabolism
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notoginseng using high-performance liquid chromatography with diode array
D. Shangguan, H. Han, R. Zhao, Y. Zhao, S. Xiong, G. Liu, New method for
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high-performance liquid chromatographic separation and fluorescence detection
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of ginsenosides, J. Chromatogr. A. 910 (2001) 367–72. S.W. Kwon, S.B. Han, I.H. Park, J.M. Kim, M.K. Park, J.H. Park, Liquid
chromatographic determination of less polar ginsenosides in processed ginseng, J. Chromatogr. A. 921 (2001) 335–339.
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X. Deng, G. Gao, S. Zheng, F. Li, Qualitative and quantitative analysis of
flavonoids in the leaves of Isatis indigatica Fort. by ultra-performance liquid chromatography with PDA and electrospray ionization tandem mass spectrometry detection, J. Pharm. Biomed. Anal. 48 (2008) 562–567.
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D.J. Chen, H.M. Liu, S.F. Xing, X.L. Piao, Cytotoxic activity of gypenosides and
cr
gynogenin against non-small cell lung carcinoma A549 cells, Bioorg. Med. Chem.
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Lett. 24 (2014) 186–191.
L. Liu, J. Huang, X Hu, K. Li, C. Sun, Simultaneous determination of ginsenoside
an
(G-Re, G-Rg1, G-Rg2, G-F1, G-Rh1) and protopanaxatriol in human plasma and urine by LC-MS/MS and its application in a pharmacokinetics study of G-Re in
M
volunteers, J. Chromatogr. B. Analyt. Technol. Biomed. Life. Sci. 879 (2011)
H.M. Liu, Y.J. Gao, X.L. Piao, Microbial transformation of gypenoside XLVI by
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Lactobacillus delbrueckii subsp. Bulgaricus, Food Science 35 (2014) 45 51. G. Chen, M. Yang, Y. Song, Z. Lu, J. Zhang, H. Huang, S. Guan, L. Wu, D.A. Guo,
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high-performance liquid chromatography coupled with tandem mass spectrometry, Biomed. Chromatogr. 22 (2008) 779–785.
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U. Kim, M.H. Park, D.H. Kim, H.H. Yoo, Metabolite profiling of ginsenoside Re
in rat urine and faeces after oral administration, Food. Chem. 136 (2013) 1364–1369. [27]
D.G. Popovich, D.D. Kitts, Structure-function relationship exists for ginsenosides in reducing cell proliferation and inducing apoptosis in the human leukemia
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Ac ce p
te
d
M
an
us
cr
ip t
(THP-1) cell line, Arch. Biochem. Biophys. 406 (2002) 1–8.
Page 18 of 31
Figure captions Figure 1. Structures of possible metabolites of G. pentaphyllum extract Figure 2. Representative MRM chromatograms for the gypenosides in serum samples
ip t
Figure 3. Representative MRM chromatograms for the gypenosides in urine samples
Figure 4. Representative MRM chromatograms for the gypenosides in faeces samples
cr
Figure 5. SIR chromatograms of blank and treated rat serum, urine, faeces samples
Ac ce p
te
d
M
an
us
Figure 6. Proposed metabolic pathways of G. pentaphyllum extract in rats
Page 19 of 31
Highlights ►A sensitive and specific UPLC/MS method was utilized. ►It can rapidly identify metabolites of Gynostemma pentaphyllum extract in rat samples. ►A total of seven
ip t
metabolites of G. Pentaphyllum extract (GP) were assigned.►Two were from the rat serum and seven were both from the rat urine and faeces. ► The proposed metabolic
Ac ce p
te
d
M
an
us
cr
pathways of GP in rats were explored.
Page 20 of 31
Figure
Ac
ce pt
ed
M
an
us
cr
ip t
Figure 1
Page 21 of 31
Ac
ce pt
ed
M
an
us
cr
ip t
Figure 2
Page 22 of 31
Ac
ce pt
ed
M
an
us
cr
ip t
Figure 3
Page 23 of 31
Ac
ce pt
ed
M
an
us
cr
ip t
Figure 4
Page 24 of 31
Ac
ce pt
ed
M
an
us
cr
ip t
Figure 5
Page 25 of 31
Ac
ce pt
ed
M
an
us
cr
ip t
Figure 6
Page 26 of 31
cr
i Compound
Linear regression equation
r
us
Linear regression, LOQs and LODs, intra-day and inter day precisions and recovery for six compounds in extract Linear
LOQ (ng)
LOD (ng)
range
(S/N
(S/N = 3)
(ng/ml)
10)
=
an
Table 1
Intra-day precision RSD (%, n=6)
Inter-day Recovery (n =6) precision Original Added RSD (%, (μg/mg) (μg) n=4)
Deteceted Recovery RSD (μg) (%) (%)
0.09
0.045
1.89
3.52
192.21
200.00
386.34
97.07
2.13
0.10
0.08
3.15
3.44
178.13
200.00
373.78
97.83
1.79
0.12
0. 05
2.71
3.05
0
0.10
0. 045
1.89
3.56
15.26
20.00
35.15
99.45
2.32
20-2000
0.16
0. 08
2.74
3.52
0
20-2000
0.18
0. 09
2.32
2.81
0
Y=0.154236X-0.4043
0.9962
20-2000
gypenoside XLVI
Y=0.147324X-1.1907
0.9992
20-2000
gypenoside L
Y=0.12199X+1.0014
0.9956
20-2000
gypenoside LI
Y=0.103574X-0.0322
0.9960
20-2000
damulin B
Y= 0.117337X+0.0189
0.9972
damulin A
Y=0.0904974X+1.1206
0.9905
Ac
ce
pt
ed
M
gypenoside LVI
Page 27 of 31 1
cr
i Compound
Linear regression equation
r
us
Linear regression, LOQs and LODs, intra-day and inter day precisions and recovery for two compounds in serum Linear
LOQ (ng)
LOD (ng)
range
(S/N
(S/N = 3)
(ng/ml)
10)
Y=0.0163657X-0.6543
0.9977
20-2000
gypenoside XLVI
Y=0.0156428X-1.4352
0.9985
20-2000
Intra-day precision RSD (%, n=6)
Inter-day Recovery (n =6) precision Original Added RSD (%, (μg) (μg) n=4)
Deteceted Recovery RSD (μg) (%) (%)
0.18
0.07
1.93
3.86
6.09
10.00
16.01
99.2
3.15
0.20
0.08
2.85
4.13
10.13
10.00
20.01.
98.8
2.73
Ac
ce
pt
ed
M
gypenoside LVI
=
an
Table 2
Page 28 of 31 2
cr
i Compound
Linear regression equation
r
us
Linear regression, LOQs and LODs, intra-day and inter day precisions and recovery for six compounds in urine Linear
LOQ (ng)
LOD (ng)
range
(S/N
(S/N = 3)
(ng/ml)
10)
=
an
Table 3
Intra-day precision RSD (%, n=6)
Inter-day Recovery (n =6) precision Original Added RSD (%, (μg) (μg) n=4)
Deteceted Recovery RSD (μg) (%) (%)
0.16
0.065
3.92
3.40
1.69
2.00
3.61
96.0
1.45
0.20
0.08
3.50
3.74
2 .70
3.00
5.65
98.33
2.26
0.14
0. 05
3.77
2.57
2.65
2.00
4.73
100.01
2.85
20-2000
0.10
0. 045
2.55
4.37
3.17
3.00
6.07
97.6
2.01
0.9989
20-2000
0.20
0. 08
2.75
4.51
2.81
3.00
5.74
97.7
1.97
0.9960
20-2000
0.19
0. 09
3.73
4.61
1.65
2.00
3.61
98.0
2.15
Y=0.0168845X+0.3293
0.9969
20-2000
gypenoside XLVI
Y=0.0147866X-1.8904
0.9986
20-2000
gypenoside L
Y=0.0672750X+0.5929
0.9913
20-2000
gypenoside LI
Y=0.0982409X-0.1259
0.9928
damulin B
Y= 0.0708798X-0.0930
damulin A
Y=0.0252250X+0.3946
Ac
ce
pt
ed
M
gypenoside LVI
Page 29 of 31 3
cr
i Compound
Linear regression equation
r
us
Linear regression, LOQs and LODs, intra-day and inter day precisions and recovery for six compounds in faeces Linear
LOQ (ng)
LOD (ng)
range
(S/N
(S/N = 3)
(ng/ml)
10)
=
an
Table 4
Intra-day precision RSD (%, n=6)
Inter-day Recovery (n =6 precision Original Added RSD (%, (μg) (μg) n=4)
Deteceted Recovery RSD (μg) (%) (%)
0.20
0.08
3.72
4.29
4.43
5.00
9.35
98.4
2.93
0.15
0.09
3.82
4.09
4.95
5.00
9.86
98.2
3.15
0.16
0. 055
2.65
4.56
3.58
5.00
8.49
98.2
3.46
20-2000
0.15
0. 065
2.84
3.66
6.33
5.00
11.19
97.2
2.86
0.9981
20-2000
0.18
0. 09
2.39
3.18
5.86
5.00
10.74
97.6
2.38
0.9981
20-2000
0.20
0. 10
2.21
2.66
1.24
2.00
3.16
96.0
1.97
Y=0.0163226X+0.9140
0.9976
20-2000
gypenoside XLVI
Y=0.0166629X-1.2393
0.9965
20-2000
gypenoside L
Y=0.0902131X-0.1186
0.9917
20-2000
gypenoside LI
Y=0.0931877X-0.1126
0.9988
damulin B
Y= 0.0664668X+0.1579
damulin A
Y=0.1056950X-0.2134
Ac
ce
pt
ed
M
gypenoside LVI
Page 30 of 31 4
i cr
Metabolites in the urine samples (ng/ml) 1
c
2
3
Mean
gypenoside LVI
67.10
94.55
23.23
61.63
gypenoside XLVI
117.40
131.79
65.97
195.05
gypenoside L
127.41
185.36
86.73
133.17
gypenoside LI
94.58
154.68
66.46
105.24
damulin B
111.00
104.25
66.25
93.83
damulin A
65.13
72.71
46.28
61.37
Metabolites in the faeces samples (ng/mg) 1
d
2
an
Gypenosides
us
Excretion of gypenoside metabolites in the serum, urine and faeces after the oral administration of GP.a
Each rat was administered on average 50 mg of GP.
b
Not detected, which is considered as 0.
c
Volume of urine sample (25.3, 20.5, 30.6 ml)
d
Weight of faeces sample (8530.2, 9380.4, 9873.5 mg)
e
Volume of serum sample (3.5, 3.8, 4.2 ml)
3
Mean
1
e
2
3
Mean
0.44
0.38
0.45
1740.01
1522.86
1615.55
1626.14
0.57
0.52
0.50
0.53
2894.36
2593.77
2696.94
2728.35
b
0.42
0.80
0.46
0.56
ND
ND
ND
ND
0.61
1.14
0.64
0.80
ND
ND
ND
ND
0.56
0.84
0.59
0.67
ND
ND
ND
ND
0.14
0.14
0.07
0.12
ND
ND
ND
ND
Ac
ce
pt
ed
a
Metabolites in the serum samples (ng/ml)
0.52
M
Table 5
Page 31 of 31 5
