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Pharmacokinetic studies of active triterpenoid saponins and the total secondary saponin from Anemone raddeana Regel
Accepted Manuscript Title: Pharmacokinetic studies of active triterpenoid saponins and the total secondary saponin from Anemone raddeana Regel Author: Dandan Zhang Tianli Lei Chongning Lv Huimin Zhao Haiyan Xu Jincai Lu PII: DOI: Reference:
S1570-0232(16)30621-3 http://dx.doi.org/doi:10.1016/j.jchromb.2017.01.003 CHROMB 20417
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
15-8-2016 7-12-2016 1-1-2017
Please cite this article as: Dandan Zhang, Tianli Lei, Chongning Lv, Huimin Zhao, Haiyan Xu, Jincai Lu, Pharmacokinetic studies of active triterpenoid saponins and the total secondary saponin from Anemone raddeana Regel, Journal of Chromatography B http://dx.doi.org/10.1016/j.jchromb.2017.01.003
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Pharmacokinetic studies of active triterpenoid saponins and the total secondary saponin from Anemone raddeana Regel
Dandan Zhanga Tianli Leia Chongning Lva Huimin Zhaob Haiyan Xub * Jincai Lua *
a
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University,
103 Wenhua Road, Shenyang 110016, China b
School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road,
Shenyang 110016, China
*Corresponding authors: Haiyan Xu Tel./Fax: +86 2423986250
E-mail address:
Jincai Lu
+86 2423986500
[email protected]
[email protected]
ABSTRACT The rhizome of Anemone raddeana Regel, a Traditional Chinese Medicine (TCM) which has a robust history treating rheumatism and neuralgia. The total secondary saponin (TSS) from it has demonstrated antitumor activity. In this study, a rapid and validated LC-MS/MS method was developed to simultaneously determine the active compounds (Hederacolchiside A1 and Eleutheroside K). Analytes were separated on a reverse-phase C18 column with acetonitrile-water (5 mmol/L ammonium acetate) as the mobile phase. This assay showed acceptable linearity (r > 0.99) over the concentration range 5-1000 nmol/L for two analytes. The intra- and inter-day precision was within 8.06% and accuracy was ranged from -3.16% to 3.34% for two analytes. The mean extraction recoveries of analytes and IS from rat plasma were all more than 76.0%. Under the developed analytical conditions, the obtained values of main pharmacokinetic parameters (Cmax and AUC0-t) indicated that the pure compounds were more efficient than the TSS extract in Hederacolchiside A1 and Eleutheroside K absorption. In addition, pharmacokinetic studies of two individual compounds demonstrated their poor oral absorption in rat (aF%, 0.019-1.521). In the study of absorption and transportation of Hederacolchiside A1 and Eleutheroside K in Caco-2 cell monolayer model, the uptake permeability was in 10-6 cm/sec range suggesting poor absorption, which confirmed the previous pharmacokinetic profiles in vivo. Interestingly, the uptake ratio of them declined significantly when treated with phloridzin (SGLT1 inhibitor). It indicated that the absorption of Hederacolchiside A1
in intestine was mainly through positive transport and SGLT1 might participate in its active absorption.
Keywords: Anemone raddeana Regel; Triterpenoid saponins; LC-MS/MS; Pharmacokinetics; Caco-2 cell monolayer model
1. Introduction Anemone raddeana Regel belonging Ranunculaceae family is widely distributed in the northeast of China, Russia (Far East), Japan and Korea [1]. The rhizome of Anemone raddeana Regel, also called “Liangtoujian”, is a Traditional Chinese Medicine (TCM) which has been officially listed in the Chinese Pharmacopeia for the treatment of rheumatism and neuralgia [2]. Extensive phytochemical and pharmacological studies on this plant proved that triterpenoid saponins were the main bioactive components of this plant [3, 4, 5, 6]. The crude saponin from Anemone raddeana Regel was demonstrated to significant antitumor activity in vitro and in vivo. It had been reported a significant inhibition on KB，HCT-8，MCF-7WT and MCF-7/ADR cell lines measured with MTT in vitro, and the 50% inhibition concentration (IC50) was 7.68, 18.52, 17.34 and 19.43 μg/mL respectively. Furthermore, obvious inhibitory effect on the growth of EAC, sarcoma S180, H22 and cervical carcinoma was observed after an oral administration with the crude saponin of “Liangtoujian” [7, 8]. Our previous pharmacological research of the total secondary saponin (TSS) prepared by alkali hydrolyzation showed significant cytotoxicity on Hep-G2, MCF-7 and A549. The pharmacological activity was
indicating that TSS possibly a potential anti-cancer medicine, however, little endeavors had been made to its pharmacokinetic studies. In our preliminary study, Hederacolchiside A1 and Eleutheroside K were isolated and identified as the major active components in TSS. Their anti-tumor mechanism of membrane-damaging had been reported in literatures [9, 10], which might be related to the anti-cancer effect of TSS. Hence, Hederacolchiside A1 and Eleutheroside K were chosen as the marker compounds for the pharmacokinetic studies of TSS. Furthermore, herbal ingredient-ingredient interactions have been reported in many medicine herbs [11-13]. Potential interactions among components in TCMs are getting more and more attention [14]. Therefore, in the present study, we not only investigated the pharmacokinetics of TSS in rats, but also compared the pharmacokinetic profiles of the active components in rats after consumptions of TSS versus pure compounds. Poor oral bioavailability have been considered as one of the general characteristics of triterpenoid saponins, which was proved by abundant of studies, such as ginsenosides, licorice saponins, astragalosides and saikosaponins [15-18]. The membrane permeability of triterpenoid saponins across Caco-2 cell monolayers revealed that the poor absorption mainly contributed to their low bioavailability [19]. However, there is still lacking of the information of intestinal absorption and transportation of Hederacolchiside A1 and Eleutheroside K.
Till now, limited analytical methods were developed for the detection of Anemone raddeana Regel [20, 21]. Several HPLC-UV methods had ever been used in the quantification of the extract of this plant. But the low sensitivity and long detection time of the HPLC-UV analysis couldn’t meet the requirement for pharmacokinetic research. LC-MS/MS is an effective and sensitive technique for compound analysis in
biosamples. Liu et al. [22] and Luan et al. [23] utilized LC-MS/MS to investigate the pharmacokinetic studies of Raddeanin A, which was another representative triterpenoid saponin in Anemone raddeana Regel. An LC-MS/MS method was established for quantification of Hederacolchiside A1 and Eleutheroside K in rat plasma after administration of extract of Pulsatilla chinensis [24]. In the reported method, solid-phase extraction (SPE) was used for sample preparation. In the present research, a sensitive and convenient LC-MS/MS with protein precipitation was developed for the measurement of Hederacolchiside A1 and Eleutheroside K in rat plasma to investigate the pharmacokinetics of TSS, the potential interaction of TSS, and the absorption mechanism of the test compounds. This information will be beneficial for understanding pharmacological and even clinical effects of such plant materials.
2. Materials and methods 2.1. Chemicals and materials The rhizome of Anemone raddeana Regel (Ranunculaceae) was purchased from AnguoTongling Medicinal Materials Co., Ltd (Place of origin: Jilin, Batch lot: 20100916), and authenticated by Prof. Jincai Lu, the School of Traditional Chinese Material Medica, Shenyang Pharmaceutical University. Two standard triterpenoid saponins (Hederacolchiside A1and Eleutheroside K) (purity > 95%) and the TSS extract were prepared in Pharmacognosy laboratory, the School of Traditional Chinese Material Medica, Shenyang Pharmaceutical University Shenyang, China. They were characterized by spectral methods, including
1
H- and
13
C-NMR
spectroscopy. The data were consistent with those reported in literature [25]. Triamcinolone acetonide (purity > 98%) used as the internal standard (IS), was purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). The structures of the compounds are shown in Fig.1. Acetonitrile (HPLC grade) was used for LC-MS/MS analysis and plasma sample preparation obtained from Dikma Technologies Inc (USA). De-ionized water was obtained from a Milli-Q water purification system (USA). All the other reagents were of analytical purity. 2.2. Animals and treatments Male Sprague-Dawley rats (n=24, 180-220 g) were provided by the Experimental Animal Center of Shenyang Pharmaceutical University (Shenyang, PR China)，animal license number: SCXK Liao 2014-0001. All animals were kept under the same laboratory conditions of temperature (25 ± 2C) and lighting (12:12 h, light:dark cycle) and were given free access to standard laboratory chow and tap water for seven days until 12 h prior to experiments. Animal experiments were performed in accordance with the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People’s Republic of China. 2.3. Preparation of the TSS extract The rhizome of Anemone raddeana Regel (Ranunculaceae) was soaked in 70% ethanol-alkali (PH=13) solution overnight, then refluxed for 1.5 h at 90C three times. The extract was subsequently dried under reduced pressure conditions. The residue was chromatographed on a resin column, and eluted with water, 20% ethanol and
80% ethanol sequentially. The fractions eluted with 80% ethanol were combined, dried in vacuum, and a powder (TSS) was obtained. 2.4. Determination of the two saponins’ contents in the TSS extract 2.4.1. HPLC-UV analytical conditions To calculate the doses to be administered, the amounts of two maker compounds in the TSS extract were quantitatively determined using HPLC-UV. Analysis was performed on a Lab PC 3000 liquid chromatography system (Lab Alliance Co., USA) connected to an EZ Chrome software. Chromatographic separation was carried on a Thermo Hypersil C18 column (250 mm × 4.6 mm, 5 μm) at 30C. UV absorption was measured at 210 nm. Gradient elution was employed using solvent A (acetonitrile) and solvent B (water); the gradient program used as follows: 0 min, A-B (40:60, v/v), 30 min, linear change to A-B (55:45, v/v). The flow rate was kept at 1.0 mL/min and the sample injection volume was 10 μL. 2.4.2. Sample preparation A portion of the dried TSS powder (10 mg) was placed into a 10 mL text tube containing 5 mL methanol. After ultrasonication for 5 min at room temperature (25C), the supernatant was transferred into a 10 mL volumetric flask which was brought up total volume with methanol, and filtered through a 0.45 μm filter prior to HPLC analysis. 2.4.3 Quantification of two marker saponins from the TSS by HPLC-UV
Representative HPLC chromatograms of standard solution and the TSS are shown in supplementary data. The contents of the two saponins in the TSS were measured quantitatively by external standard method by HPLC-UV. The contents of Hederacolchiside A1 and Eleutheroside K in total saponins were (43.0 ± 0.51) % and (5.62 ± 0.03) %, respectively. 2.5. LC-MS/MS analytical conditions All separations were performed on the Agilent 1290 Infinity Rapid Resolution Liquid Chromatography System equipped with a vacuum degasser, a binary pump and an autosampler. Chromatographic separation was achieved on an Agela C18 columnn (4.6×150 mm, 5 m) (Bonna-Agela Technologies Inc.). The column temperature was maintained at 30C, and the injection volume was 10 μL. The temperature of autosampler was at 4C. A gradient mobile phase consisting of solvent A, acetonitrile, and solvent B, 5 mmol/L ammonium acetate was delivered at 1 mL/min and the split ratio was 1:1. Elution conditions were as follows: 0-2.8 min, 60% A; 2.8-2.9 min, 60%-80% A; 2.9-3.5 min, 80% A; 3.5-3.6 min, 80%-60% A; 3.6-5 min, 60% A. The total run time was 5 min. MS analysis was performed on an API 4000 triple quadrupole mass spectrometer equipped with Turbo ion spray sources (TIS sources). The TIS source parameters were as follows: ion spray (IS) voltage: -4000 V; temperature (TEM): 600C; Gas 1 and Gas 2 were set at 50 and 60 psi, respectively. The curtain gas and collision gas were set at 15 and 6 psi, respectively. Quantitative analysis was performed using the multiple-reaction monitoring (MRM) mode. The precursor-to-production pair and compound-dependent parameters, viz. declustering
potential (DP) and collision energy (CE) were optimized for each analyte and selected values are listed in supplementary data. The dwell time for each MRM transition was automatically set at 80 ms by the software. All instrumentation was controlled and synchronized by Analyst software (versions 1.6.) from Applied Biosystems/MDS Sciex. 2.6. Method validation of LC-MS/MS 2.6.1. Preparation of standard solution and quality control samples Standard stock solutions were prepared by dissolving two accurately weighed standards in methanol, two standards (Hederacolchiside A1and Eleutheroside K) were at the same concentration of 500 μmol/mL. A series of working solutions of two analytes were prepared by diluting the mixed standard solutions with methanol in appropriate ratios to yield concentrations of 5-1000 nM for calibration curves and quality control (QC) samples. A solution for internal standard (2 μg/mL) was prepared in methanol. All solutions were stored at -4C. Calibration curves were prepared by spiking with 10 µL different concentrations of the standard working stock solutions to 50 µL blank rat plasma sample subsequently. Calibration curves were prepared at concentrations of 5, 10, 50, 100, 250, 500, 1000 nM for plasma. The similar method was also used to prepare QC samples (namely LLOQ, low QC, med QC and high QC) in rat plasma at concentrations of 5, 20, 200 and 800 nM. 2.6.2. Preparation of plasma samples
An aliquots of 50 μL of rat plasma were spiked with 10 μL of IS solution (2 g/mL) and then precipitate agent of 100 μL acetonitrile was added. The sample had been vortex for 50 s, and centrifuged at 13,000 rpm for 5 min. The supernatant was evaporated to dryness under N2 for about 20 min at 45C. The residues were reconstituted in 100 μL and were centrifuged at 15,000 rpm for 10 min. An aliquot of 10 μL of the final testing samples was injected onto the LC-MS/MS system for analysis. 2.6.3. Method validation The method was validated in terms of specificity, selectivity, calibration curve, sensitivity, matrix effect, accuracy, precision and stability, in accordance with the USA Food and Drug Administration (FDA) bioanalytical method validation guidance [26]. The calibration was performed by least-squares linear regression of the ratio of peak area of each analyte to the IS vs the nominal standard concentration with a weighted factor (1/x). The LLOQ was defined as a reproducible lowest possible concentration, linear with the calibration curves having a relative error between -20% and 20%. To assess the intra-day accuracy and precision of the method, three concentrations levels of QC solution were spiked into plasma, with 6 replicates at each concentration. The extraction recoveries were determined by comparing the peak area obtained from plasma samples spiked before extraction with those from plasma samples spiked after extraction. The matrix effects were evaluated by comparing the peak areas obtained from samples where the extracted matrix was spiked with standard solutions to those obtained from the pure reference standard solutions at the
same concentration. Stability of QC samples was assessed by analyzing samples stored at -70C for 1 month, subjected to three freeze thaw (room temperature) cycles, and stored at room temperature for 24 h. Samples were considered to be stable if the deviation from the nominal concentration was within ± 15%. 2.7. Pharmacokinetic studies of active triterpenoid saponins and the TSS extract On account of the poor solubility in water, Hederacolchiside A1, Eleutheroside K and the TSS extract were suspended separately in an aqueous solution of 0.5% sodium carboxymethyl cellulose (CMC-Na) to get an evenly dispersed suspension for oral administration. For intravenous group, Hederacolchiside A1 and Eleutheroside K were dissolved in (1% DMSO) 0.9% injectable NaCl solution respectively. Eighteen rats were randomly assigned to 6 groups (three rats per group) to be orally administration. In the result: rats in two groups were orally administrated with Hederacolchiside A1 at two dosages of 20 μmol/kg (17.93 mg/kg)and 200 μmol/kg (179.3 mg/kg); two groups were with Eleutheroside K at oral doses of 2 μmol/kg (1.469 mg/kg) and 20 μmol/kg (14.69 mg/kg). The last two groups were oral administrated with the TSS at two oral doses (41.7 and 417.0 mg/kg). It should be noted that the oral dosage (41.7 mg/kg) of the TSS to rats was calculated from the effective dose of Hederacolchiside A1 (20 μmol/kg) while the other oral dosage (417.0 mg/kg) was based on the effective dose of Eleutheroside K (20 μmol/kg). Blood samples (150 μL) were collected from the oculi chorioideae vein of each rat subsequently at 0, 0.33, 0.67, 1, 1.5, 2, 4, 8, 12, 24, 36, 48 h after oral administration.
The collected blood samples were placed in heparinized tubes and separated by centrifugation at 4000 rpm for 5 min. The supernatant was transferred into 1.5 mL eppendorf tubes and stored at -70C prior to analysis. To obtained the value of absolute bioavailability and more pharmacokinetic properties of two makers in rats. Another 2 groups of rats (three rats per group) were administered
an
intravenous
dose
at
0.5
μmol/kg
of
two
components
(Hederacolchiside A1 and Eleutheroside K) separately. Blood samples (150 μL) were collected from the oculi chorioidea vein of each rat subsequently at 0, 0.083, 0.17, 0.5, 1, 2，4, 6, 8, 12, 24, 36 h after intravenous administration. The collected blood samples were conducted in the same manner as above. The animal experiment procedure was approved by the Animal Ethics Board of Shenyang Pharmaceutical University. 2.8. Transport experiments in the Caco-2 cell culture model The Caco-2 cells were used at passages 20-30. The transcellular transport study was performed as described previously [27]. DMSO in DMEM (Dulbecco’s modified Eagle’s medium, 0.1% (v/v), Gibco Invitrogen, Carlsbad, CA) was used as control. After the cells in the flask grew to 90-100% confluency, cells were trypsinized and seeded on collagen-coated polyte-trafluroethylene membrane inserts (0.45 μm) fitted in bicameral chambers (Transwell-COL, 24 mm ID, Corning Inc., Corning, NY) at 1.2 × 105 cells/cm2. The transepithelial electrical resistance (TEER) was tested by Millicell ERS meter (Fisher Sci., Pittsburgh, PA) to reflect the tightness of intercellular junctions and only cells with TEER ≥ 250 Ωcm2 were used for permeability study. Hederacolchiside A1 and Eleutheroside K solution at was loaded
onto the apical or basolateral (donor) side. Three donor samples (500 μL) and three receiver samples (500 μL) were taken at 0, 1, 2, 3, and 4 h followed by the addition of 500 µL of fresh donor solution to the donor side or 500 µL of fresh buffer to the receiver side. The donor side concentrations of the test compounds were always10 µmol/L ether from apical to basolateral or basolateral to apical directions. In the present study, inhibitor (phloridzin 20 µg/mL) of uptake transporter was only added to the apical side of cell monolayers, regardless of where test compounds were loaded. The samples were then analyzed by LC-MS/MS.
2.9. Statistical methods Experimental data and pharmacokinetic parameters are expressed as means ± SD. Pharmacokinetic parameters were calculated by the PK analysis software DAS 2.0 (Chinese Pharmacological Society) using the non-compartmental method. The Cmax and Tmax were observed values without interpolation. The area under the concentration-time curve up to the last measured time point (AUC0-t) was calculated using the trapezoidal rule. The value of AUC0- ∞ was generated by extrapolating AUC0-t to infinity using the elimination rate constant (ke) and the last measured concentration. Apparent elimination half-life (T1/2) was calculated as T1/2 = 0.693/ke, total body clearance (CL) as dose/ AUC0-∞, and apparent volume of distribution (V) as CL/ke. To compare parameters between herb extract group and monomer group, statistical significances were evaluated by Student’s t-tests with computer software SPSS.P value < 0.05 was considered statistically significant. The apparent permeability coefficient (P) was determined by the Equation P = (dQ/dt) A × C0 where dQ/dt is the drug permeation rate (mol/s), A is the surface area of the
epithelium (cm2), and C0 is the initial concentration in the donor compartment at time 0 (nM). Both permeability from the apical to basolateral side (Pab) and basolateral to apical side (Pba) were calculated according to the above equation. The uptake ratio was calculated as Pab/Pba, which represented the degrees of uptake transporter involvement.
3. Results and discussion 3.1. Method development of LC-MS/MS To investigate the pharmacokinetic profiles, it was necessary to develop a rapid, sensitive and robust method for the determination of two triterpenoid saponins in rat plasma. An LC method coupled with tandem mass spectroscopy was developed to achieve this goal. The chromatographic conditions, especially analytical columns and mobile phase compositions (concentration of buffer and percentage of the organic modifiers), were optimized through trials and errors to obtain symmetric peak shape and short run time for the simultaneous analysis of the two compounds. It was observed that acetonitrile gave a better peak shape than methanol, and therefore was selected as the organic phase in mobile phase. Compared with water, the addition of ammonium acetate as a component of the mobile phase was found to make the peak shaper, improve sensitivity and shorten retention time of each analyte. Under the developed chromatographic conditions for simultaneous determination of the two compounds, all analytes were eluted rapidly within 4.0 min. Recent studies have reported the use of ESI-MS for determination of triterpenoid saponins with higher sensitivity and better reproducibility than the other types of ionization [28-30]. In the investigation, both positive and negative modes of
ionization were tested. The response and sensitivity observed in the negative ion mode were higher than that in positive ionization mode. In negative ionization mode, the analytes formed deprotein ions [M−H]−; however, IS produced adduct ion [M+CH3COO]− with acetic acid. Generally, the MS2 fragmentation pathways of saponins were similar involving the loss of various sugar moieties without any parent-ring cleavage which are shown in Fig. 1
3.2. Method validation of LC-MS/MS The chromatogram of a blank plasma sample showed no endogenous peaks interference the analysis of Hederacolchiside A1 and Eleutheroside K. Due to the efficient sample treatment and high selectivity of MRM, matrix effect for these analytes was insignificant. Typical chromatograms of blanks, spiked plasma, along with rat plasma sample after i.g. and i.v. administration are shown in Fig. 2. The calibration curve of Hederacolchiside A1 was y = 0.000208x + 0.00043 (r=0.9969) and y = 0.000898x + 0.00106 (r=0.9995) was for Eleutheroside K. The LLOQs for Hederacolchiside A1 and Eleutheroside K were 5 nM, with accuracy and precision no higher than ± 20% RE and 20% RSD. Table 1 summarized the intra- and the inter-day precision and accuracy of the method. The intra-day and inter-day precisions ranged from 0.73% to 8.06% and 0.73% to 7.88% for both analytes, respectively. The accuracy derived from QC samples was in the range from -3.16% to 3.34%. The result indicated that the assay had good reproducibility with acceptable accuracy and precision.
As shown in Table 2, the mean extraction recoveries of all analytes at different concentrations were from 76.0 to 87.8%. The extraction recovery of IS was 91.0 ± 6.9%. The matrix effect of blank plasma of the analytes and IS was found to be within the acceptable range, suggesting that the plasma matrix effect was negligible for the assay. Stability of the analytes during the sample storing and processing procedures was fully evaluated by analysis of corresponding QC samples. Results of the stability tests shown in Table 3 showed that both of the analytes were stable in plasma samples for 1 month at -70C, 24 h at room temperature (4C) and three freeze-thaw cycles.
3.3. Pharmacokinetic behavior comparison between the consumptions of the TSS versus pure monomers in rats
The plasma concentrations of Hederacolchiside A1 and Eleutheroside K were determined by the established LC-MS/MS method after rats were orally given the TSS extract and the pure monomers at two doses. The mean plasma concentration-time curves of Hederacolchiside A1 and Eleutheroside K after oral dosage are presented in Fig. 3, respectively. The concentration-time curves of both Hederacolchiside A1 and Eleutheroside K had two- or three-peak after oral administration, which might be caused by enteric circulation. This phenomenon is usually observed in the pharmacokinetic study of herbal medicines [31, 32].
The major pharmacokinetic parameters of Hederacolchiside A1 after oral administration of TSS and pure compound calculated by DAS 2.0 software are listed in Table 4. After oral administration of TSS, Hederacolchiside A1 reached the maximum concentration (Cmax) of (41.77 ± 10.90) nmol/L and (80.00 ± 14.64) nmol/L at the doses of 41.7 and 417.0 mg/kg, respectively. The area under curve of the concentration-time profile (AUC0-t) were (209.85 ± 26.08) nmol·h/L at the dose of
41.7 mg/kg and (694.17 ± 82.03) nmol·h/L following the dose of 417.0 mg/kg. However, the values of Cmax and AUC0-t increased significantly after the equivalent oral doses of Hederacolchiside A1 monomer. The Cmax value of Hederacolchiside A1 after dosage of monomer at 20 μmol/kg (17.93 mg/kg) and 200 μmol/kg (179.3 mg/kg) was (90.69 ± 26.96) nmol/L and (1069 ± 304.77) nmol/L, respectively. The correspondent AUC0-t of Hederacolchiside A1 were (378.21 ± 58.89) nmol·h/L and (112104.96 ± 1727.08) nmol·h/L, respectively. The time of peak concentration (Tmax), and half-life (T1/2) of Hederacolchiside A1 after oral administration of TSS and pure compound did not show significant differences.
Similar to Hederacolchiside A1, the Cmax and AUC0-t data of Eleutheroside K after TSS (417.0 mg/kg) administration were statistically lower than those after administration of equivalent dose of pure monomer 20 μmol/kg (14.69 mg/kg). When rats were orally treated with TSS at 417.0 mg/kg, the Cmax of Eleutheroside K was (15.45 ± 5.59) nmol/L and the AUC0-t was (69.40 ± 9.39) nmol·h/L. As shown in Table 5, when rats were given an oral dose of 20 μmol/kg (14.69 mg/kg) pure monomer, the Cmax and AUC0-t values of Eleutheroside K were (48.13 ± 14.87) nmol/L and (136.89 ± 37.67) nmol·h/L. However, after rats were orally admistrated with lower dosage of TSS at 41.7 mg/kg or Eleutheroside K monomer at 2 μmol/kg (1.469 mg/kg), there were no significant differences in the parameters between the two groups.
The results indicated that the pure compounds were more efficient than the TSS extract in the absorption of Hederacolchiside A1 and Eleutheroside K. The present
study was the first to report the difference existing in pharmacokinetic profiles of the test active compounds administrated in herb extract and monomer. Zeng et al. reported that the oral bioavailability of pure baicalin was higher than that of the herbal preparation of Huang-Lian-Jie-Du-Tang, a formulation containing Rhizoma Coptidis, Radix Scutellariae, Cortex Phellodendri and Fructus Gardenia [33]. They illuminated that the lower oral bioavailability of baicalin after administration of herbal preparation was resulted from the inhibited absorption of baicalin by berberine, a quaternary ammonium compound [34]. The mechanisms of the worse absorption of Hederacolchiside A1 and Eleutheroside K following dose of TSS were not investigated in the present study. We speculated that some ingredients in TSS extract weaken the absorption of both active ingredients. Further studies are scheduled to elucidate the underlying mechanism of the pharmacokinetic differences.
In the present study, we also conducted the i.v. pharmacokinetics of pure Hederacolchiside A1 and Elutheroside K to study the absolutely bioavailability. The concentration-time curves of Hederacolchiside A1 and Eleutheroside K after i.v. administration are shown in Fig. 3. Elutheroside K exhibited rapid elimination while Hederacolchiside A1was moderate after i.v. injection, which was also observed after oral administration. In the previous literatures, most of saponins were susceptible to rapid and extensive biliary excretion through active transport, which may lead to short T1/2, low systemic exposure [35]. It’s valuable that Hederacolchiside A1 such a long-circulating saponin used as pharmacokinetic marker to substantiate systemic exposure to the ingested herb extract. Furthermore, the absolute bioavailability of
pure Elutheroside K was 0.30% and the value of Hederacolchiside A1 was 0.141% at the same oral dosage. The low oral bioavailability of the two triterpenoid saponins might be attributed to their poor gastrointestinal absorption [36,37], because their molecular masses (733.5 Da and 896.5 Da) were larger than the favorable value (<500 Da) for membrane permeability [38]. Further studies were performed to investigate the absorption mechanism of the two pure compounds.
3.4. Absorption mechanism in the Caco-2 cell The absorption permeability of Hederacolchiside A1 (3.09 ± 0.33×10-6 cm/sec) was significantly higher than that of efflux permeability (0.61 ± 0.20×10 -6 cm/sec). Notably, the uptake ratio of 5.03 indicated that uptake transporter(s) was involved in the absorption of Hederacolchiside A1. However, the uptake ratio was declined to 2.03 when phloridzin, a SGLT1 inhibitor, was added into the apical side, suggesting that SGLT1 probably participate in the active absorption of Hederacolchiside A1. The transporter behavior of Eleutheroside K in the Caco-2 cell culture model was tested in a similar method to that of Hederacolchiside A1. When (SGLT1 inhibitor) phloridzin exist, the uptake permeability decreased significantly (from 2.87 ± 0.51×10-06 cm/sec to 1.76 ± 0.31×10-06 cm/sec) while the efflux permeability was (from 1.52 ± 0.45×10-6 cm/sec to 1.26 ± 0.78×10-6 cm/sec) (Fig. 4). SGLT1 also influenced the transport of Eleutheroside K.
The Caco-2 cell monolayer system was a standard convenient model to study human intestinal absorption of xenobiotic compounds [39]. The relatively low permeability values of about 10-6 cm/sec for Hederacolchiside A1 and Eleutheroside K illuminated their weak permeation across the intestinal tract, which was agreed with their poor
oral bioavailability. In addition, the transporter proteins involved in the active influx of both saponins were identified. The transporter SGLT1 present in the apical membrane of the small intestine seemed participating in their active absorption [40, 41]. The pharmacokinetic (PK) and absorption studies of the two active compounds discussed herein will be more productive for the exploration of TSS’s pharmacological activity.
Conclusion
A reliable LC-MS/MS method was developed for the simultaneous determination of Hederacolchiside A1and Eleutheroside K in rat plasma. It was successfully applied to the comparative pharmacokinetic studies between consumptions of the TSS versus pure monomers in rats. The result of the present study demonstrated that both Hederacolchiside A1 and Eleutheroside K showed poor absorption after oral administration and TSS matrix inhibited their absorption. In addition, Transport experiments in the Caco-2 cell culture model showed low permeability (Pab) of Hederacolchiside A1 and Eleutheroside K. Absorption of Hederacolchiside A1 was mainly through positive transport and SGLT1 might participate in its active absorption.
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Fig.1. Product ion spectra and monitored transitions of analytes.
Fig.2. Representative extract ion MRM chromatograms of two marker compounds and Triamcinolone acetonide (IS): (A) blank plasma, (B) blank plasma spiked with the two analytes at LLOQ and IS, (C) 2 h sample plasma after a single oral administration of the TSS extract at 417.39 mg/kg.
B
A
C D
.
E
F
Fig.3. Mean plasma concentration vs time profile of Hederacolchiside A1 and Eleutheroside K: (A) after oral administration of TSS extract (41.7 and 417.0 mg/kg), (B) after oral administration of Hederacolchiside A1 (20 μmol/kg and 200 μmol/kg), (C) after oral administration of TSS extract (41.7 and 417.0 mg/kg), (D) after oral administration of Eleutheroside K (2 μmol/kg and 20 μmol/kg), (mean ± SD, n = 3), (E)after intravenous administration of
Hederacolchiside A1 to rats (0. 5 μmol/kg), (F)after
intravenous administration of Eleutheroside K to rats (0. 5 μmol/kg) (mean ± SD, n = 3).
Fig.4. Transport of Hederacolchiside A1and Eleutheroside K in the Caco-2 cell culture model. The donor side concentrations of the test compounds were always 10 µmol/L either from apical to basolateral or basolateral to apical directions while phloridzin (20 µg/mL) in inhibitor group. Each data point is the average of three determinations. Bars are standard deviations of the mean. The asterisk (*) indicates P < 0.05 when A→B group compared with the corresponding B→A group of compounds.
Table 1 Precision and accuracy of two marker compounds for LC-MS/MS method (n = 6). Spiked Components (nM) Hederacolchiside A1
Eleutheroside K
Concentration
Intra-day
Inter-day
Measured
Precision
Precision
(nM)
(RSD, %)
(RSD, %)
Accuracy
(RE, %)
5
4.96 ± 0.22
1.11
1.25
-0.83
20
19.67 ± 1.10
5.24
5.27
-1.65
200
194.06 ± 11.30
6.19
0.92
-2.90
800
789.62 ± 61.40
8.06
1.72
-1.30
5
4.96 ± 0.15
0.73
0.73
-0.85
20
20.67 ± 0.99
4.24
7.88
3.34
200
197.63 ± 11.83
5.74
7.60
-1.18
800
774.69 ± 35.97
4.74
2.41
-3.16
Table 2 Recoveries and matrix effects of two marker compounds for LC-MS/MS method (n = 6). Components
Spiked (nM)
Extraction recovery (%)
Matrix effect (%)
Hederacolchiside A1
20
87.8 ± 11.5
102.7 ± 11.7
200
76.0 ± 5.6
101.4 ± 7.9
800
76.1 ± 8.0
99.5 ± 7.7
20
77.2 ± 9.1
100.4 ± 12.3
200
78.2 ± 3.1
100.6 ± 8.1
800
76.2 ± 3.4
95.7 ± 12.2
91.0 ± 6.9
101.7 ± 7.2
Eleutheroside K
IS
Table 3 Stability for analysis of two marker compounds in rat plasma (n = 6) Spiked
Short term (25°C, 24 h)
Long term (-25°C, 30 days)
Freeze-thaw
(nM)
RSD (%)
RE (%)
RSD (%)
RE (%)
RSD (%)
RE (%)
20
2.7
-2.85
2.78
-1.03
3.8
-1.33
800
4.43
7.49
4.1
7.71
4.49
1.625
20
5.18
-0.67
4.5
0.17
4.35
9.17
800
1.66
-5.88
3.84
-8
3.86
-4.46
Components
Hederacolchiside A1
Eleutheroside K
Table 4 Pharmacokinetic parameters of Hederacolchiside A1 after single oral administration of TSS extract at doses of 41.7 and 417.0 mg/kg, intraveneous administration of pure monomer at 0.5 μmol/kg and single oral administration of pure monomer at 20 and 200 μmol/kg to male rats (mean ± SD).
Component
Oral
Intraveneous
Oral
TSS
Monomer
Monomer
41.7 mg/kg
417.0 mg/kg
0.5 μmol/kg
Cmax (nmol/L)
41.77 ± 10.90
80.00 ± 14.64
AUC0-t (nmol·h/L )
209.85 ± 26.08
694.17 ± 82.03
Tmax (h)
3.33 ± 1.15
2.78 ± 2.12
T1/2 (h)
6.68 ± 6.84
15.86 ± 4.54
AUC0-∞ (nmol ·h/L)
263.93 ± 32.12
819.85 ± 141.61
20
μmol/kg
200
μmol/kg
(17.93 mg/kg)
(179.3 mg/kg)
9331.33 ± 2025.51
90.69 ± 26.96*
1069.00 ± 304.77**
12059.48 ± 3741.98
378.21 ± 58.89*
112104.96 ± 1727.08**
3.17 ± 2.46
10.00 ± 3.46
8.80 ± 1.31
5.96 ± 2.93
6.90 ± 2.44
12274.76 ± 3763.37
688.42 ± 330.59
12264.77 ± 1742.96
0.141 ± 0.068
0.251 ± 0.036
Hederacolchiside A1
V (L/Kg)
0.023 ± 0.010
CL (L/h/Kg)
0.002 ± 0.001
a
F (%)
0.054 ± 0.007
0.017 ± 0.003
The oral administration of TSS extract at 41.7 and 417.0 mg/kg was equivalent to the oral administration of Hederacolchiside A1at 20 μmol/kg and 200 μmol/kg. **P < 0.01 when compared with the corresponding group of TSS extract, *P < 0.05 when compared with the corresponding group of TSS extract. a
F (%) = [(Doseiv × AUC0–∞oral)/(Doseoral × AUC0–∞iv)] × 100
Table 5 Pharmacokinetic parameters of Eleutheroside K after single oral administration of TSS extract at doses of 41.7 and 417.0 mg/kg, intraveneous administration of pure monomer at 0.5 μmol/kg and single oral administration of pure monomer at 2 and 20 μmol/kg to male rats (mean ± SD).
Component
Oral
Intraveneous
Oral
TSS
Monomer
Monomer 2 μmol/kg
41.7 mg/kg
417.0 mg/kg
0.5 μmol/kg (1.469mg/kg)
20
μmol/kg
(14.69 mg/kg)
Cmax (nmol/L)
12.25± 4.34
15.45 ± 5.59
5233.33 ± 841.27
12.70 ± 2.19
48.13 ± 14.87*
AUC0-t (nmol ·h/L)
53.82 ±28.03
69.40 ± 9.39
1597.39 ± 90.89
40.21 ± 12.15
136.89 ± 37.67*
Tmax (h)
2.75 ± 1.77
5.00 ± 3.61
4.78 ± 3.98
1.50 ± 0.50
T1/2 (h)
4.51 ± 4.11
8.94 ± 1.71
0.8 0 ± 0.40
3.93 ± 2.84
1.74 ± 0.57
AUC0-∞ (nmol·h/L)
81.77 ± 9.38
196.85 ± 16.42
1601.77 ± 84.39
97.03 ± 3.41
191.35 ± 95.32
1.521 ± 0.053
0.300 ± 0.149
Eleutheroside K
V (L/Kg)
0.014 ± 0.008
CL (L/h/Kg)
0.012 ± 0.001
a
F (%)
1.282 ± 0.147
0.309 ± 0.026
The oral administration of TSS extract at 41.7 and 417.0 mg/kg was equivalent to the oral administration of Eleutheroside K at 2 μmol/kg and 20 μmol/kg. *P < 0.05 when compared with the corresponding group of TSS extract. a
F (%) = [(Doseiv × AUC0–∞oral)/(Doseoral × AUC0–∞iv)] × 100
S1570-0232(16)30621-3 http://dx.doi.org/doi:10.1016/j.jchromb.2017.01.003 CHROMB 20417
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
15-8-2016 7-12-2016 1-1-2017
Please cite this article as: Dandan Zhang, Tianli Lei, Chongning Lv, Huimin Zhao, Haiyan Xu, Jincai Lu, Pharmacokinetic studies of active triterpenoid saponins and the total secondary saponin from Anemone raddeana Regel, Journal of Chromatography B http://dx.doi.org/10.1016/j.jchromb.2017.01.003
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Pharmacokinetic studies of active triterpenoid saponins and the total secondary saponin from Anemone raddeana Regel
Dandan Zhanga Tianli Leia Chongning Lva Huimin Zhaob Haiyan Xub * Jincai Lua *
a
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University,
103 Wenhua Road, Shenyang 110016, China b
School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road,
Shenyang 110016, China
*Corresponding authors: Haiyan Xu Tel./Fax: +86 2423986250
E-mail address:
Jincai Lu
+86 2423986500
[email protected]
[email protected]
ABSTRACT The rhizome of Anemone raddeana Regel, a Traditional Chinese Medicine (TCM) which has a robust history treating rheumatism and neuralgia. The total secondary saponin (TSS) from it has demonstrated antitumor activity. In this study, a rapid and validated LC-MS/MS method was developed to simultaneously determine the active compounds (Hederacolchiside A1 and Eleutheroside K). Analytes were separated on a reverse-phase C18 column with acetonitrile-water (5 mmol/L ammonium acetate) as the mobile phase. This assay showed acceptable linearity (r > 0.99) over the concentration range 5-1000 nmol/L for two analytes. The intra- and inter-day precision was within 8.06% and accuracy was ranged from -3.16% to 3.34% for two analytes. The mean extraction recoveries of analytes and IS from rat plasma were all more than 76.0%. Under the developed analytical conditions, the obtained values of main pharmacokinetic parameters (Cmax and AUC0-t) indicated that the pure compounds were more efficient than the TSS extract in Hederacolchiside A1 and Eleutheroside K absorption. In addition, pharmacokinetic studies of two individual compounds demonstrated their poor oral absorption in rat (aF%, 0.019-1.521). In the study of absorption and transportation of Hederacolchiside A1 and Eleutheroside K in Caco-2 cell monolayer model, the uptake permeability was in 10-6 cm/sec range suggesting poor absorption, which confirmed the previous pharmacokinetic profiles in vivo. Interestingly, the uptake ratio of them declined significantly when treated with phloridzin (SGLT1 inhibitor). It indicated that the absorption of Hederacolchiside A1
in intestine was mainly through positive transport and SGLT1 might participate in its active absorption.
Keywords: Anemone raddeana Regel; Triterpenoid saponins; LC-MS/MS; Pharmacokinetics; Caco-2 cell monolayer model
1. Introduction Anemone raddeana Regel belonging Ranunculaceae family is widely distributed in the northeast of China, Russia (Far East), Japan and Korea [1]. The rhizome of Anemone raddeana Regel, also called “Liangtoujian”, is a Traditional Chinese Medicine (TCM) which has been officially listed in the Chinese Pharmacopeia for the treatment of rheumatism and neuralgia [2]. Extensive phytochemical and pharmacological studies on this plant proved that triterpenoid saponins were the main bioactive components of this plant [3, 4, 5, 6]. The crude saponin from Anemone raddeana Regel was demonstrated to significant antitumor activity in vitro and in vivo. It had been reported a significant inhibition on KB，HCT-8，MCF-7WT and MCF-7/ADR cell lines measured with MTT in vitro, and the 50% inhibition concentration (IC50) was 7.68, 18.52, 17.34 and 19.43 μg/mL respectively. Furthermore, obvious inhibitory effect on the growth of EAC, sarcoma S180, H22 and cervical carcinoma was observed after an oral administration with the crude saponin of “Liangtoujian” [7, 8]. Our previous pharmacological research of the total secondary saponin (TSS) prepared by alkali hydrolyzation showed significant cytotoxicity on Hep-G2, MCF-7 and A549. The pharmacological activity was
indicating that TSS possibly a potential anti-cancer medicine, however, little endeavors had been made to its pharmacokinetic studies. In our preliminary study, Hederacolchiside A1 and Eleutheroside K were isolated and identified as the major active components in TSS. Their anti-tumor mechanism of membrane-damaging had been reported in literatures [9, 10], which might be related to the anti-cancer effect of TSS. Hence, Hederacolchiside A1 and Eleutheroside K were chosen as the marker compounds for the pharmacokinetic studies of TSS. Furthermore, herbal ingredient-ingredient interactions have been reported in many medicine herbs [11-13]. Potential interactions among components in TCMs are getting more and more attention [14]. Therefore, in the present study, we not only investigated the pharmacokinetics of TSS in rats, but also compared the pharmacokinetic profiles of the active components in rats after consumptions of TSS versus pure compounds. Poor oral bioavailability have been considered as one of the general characteristics of triterpenoid saponins, which was proved by abundant of studies, such as ginsenosides, licorice saponins, astragalosides and saikosaponins [15-18]. The membrane permeability of triterpenoid saponins across Caco-2 cell monolayers revealed that the poor absorption mainly contributed to their low bioavailability [19]. However, there is still lacking of the information of intestinal absorption and transportation of Hederacolchiside A1 and Eleutheroside K.
Till now, limited analytical methods were developed for the detection of Anemone raddeana Regel [20, 21]. Several HPLC-UV methods had ever been used in the quantification of the extract of this plant. But the low sensitivity and long detection time of the HPLC-UV analysis couldn’t meet the requirement for pharmacokinetic research. LC-MS/MS is an effective and sensitive technique for compound analysis in
biosamples. Liu et al. [22] and Luan et al. [23] utilized LC-MS/MS to investigate the pharmacokinetic studies of Raddeanin A, which was another representative triterpenoid saponin in Anemone raddeana Regel. An LC-MS/MS method was established for quantification of Hederacolchiside A1 and Eleutheroside K in rat plasma after administration of extract of Pulsatilla chinensis [24]. In the reported method, solid-phase extraction (SPE) was used for sample preparation. In the present research, a sensitive and convenient LC-MS/MS with protein precipitation was developed for the measurement of Hederacolchiside A1 and Eleutheroside K in rat plasma to investigate the pharmacokinetics of TSS, the potential interaction of TSS, and the absorption mechanism of the test compounds. This information will be beneficial for understanding pharmacological and even clinical effects of such plant materials.
2. Materials and methods 2.1. Chemicals and materials The rhizome of Anemone raddeana Regel (Ranunculaceae) was purchased from AnguoTongling Medicinal Materials Co., Ltd (Place of origin: Jilin, Batch lot: 20100916), and authenticated by Prof. Jincai Lu, the School of Traditional Chinese Material Medica, Shenyang Pharmaceutical University. Two standard triterpenoid saponins (Hederacolchiside A1and Eleutheroside K) (purity > 95%) and the TSS extract were prepared in Pharmacognosy laboratory, the School of Traditional Chinese Material Medica, Shenyang Pharmaceutical University Shenyang, China. They were characterized by spectral methods, including
1
H- and
13
C-NMR
spectroscopy. The data were consistent with those reported in literature [25]. Triamcinolone acetonide (purity > 98%) used as the internal standard (IS), was purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). The structures of the compounds are shown in Fig.1. Acetonitrile (HPLC grade) was used for LC-MS/MS analysis and plasma sample preparation obtained from Dikma Technologies Inc (USA). De-ionized water was obtained from a Milli-Q water purification system (USA). All the other reagents were of analytical purity. 2.2. Animals and treatments Male Sprague-Dawley rats (n=24, 180-220 g) were provided by the Experimental Animal Center of Shenyang Pharmaceutical University (Shenyang, PR China)，animal license number: SCXK Liao 2014-0001. All animals were kept under the same laboratory conditions of temperature (25 ± 2C) and lighting (12:12 h, light:dark cycle) and were given free access to standard laboratory chow and tap water for seven days until 12 h prior to experiments. Animal experiments were performed in accordance with the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People’s Republic of China. 2.3. Preparation of the TSS extract The rhizome of Anemone raddeana Regel (Ranunculaceae) was soaked in 70% ethanol-alkali (PH=13) solution overnight, then refluxed for 1.5 h at 90C three times. The extract was subsequently dried under reduced pressure conditions. The residue was chromatographed on a resin column, and eluted with water, 20% ethanol and
80% ethanol sequentially. The fractions eluted with 80% ethanol were combined, dried in vacuum, and a powder (TSS) was obtained. 2.4. Determination of the two saponins’ contents in the TSS extract 2.4.1. HPLC-UV analytical conditions To calculate the doses to be administered, the amounts of two maker compounds in the TSS extract were quantitatively determined using HPLC-UV. Analysis was performed on a Lab PC 3000 liquid chromatography system (Lab Alliance Co., USA) connected to an EZ Chrome software. Chromatographic separation was carried on a Thermo Hypersil C18 column (250 mm × 4.6 mm, 5 μm) at 30C. UV absorption was measured at 210 nm. Gradient elution was employed using solvent A (acetonitrile) and solvent B (water); the gradient program used as follows: 0 min, A-B (40:60, v/v), 30 min, linear change to A-B (55:45, v/v). The flow rate was kept at 1.0 mL/min and the sample injection volume was 10 μL. 2.4.2. Sample preparation A portion of the dried TSS powder (10 mg) was placed into a 10 mL text tube containing 5 mL methanol. After ultrasonication for 5 min at room temperature (25C), the supernatant was transferred into a 10 mL volumetric flask which was brought up total volume with methanol, and filtered through a 0.45 μm filter prior to HPLC analysis. 2.4.3 Quantification of two marker saponins from the TSS by HPLC-UV
Representative HPLC chromatograms of standard solution and the TSS are shown in supplementary data. The contents of the two saponins in the TSS were measured quantitatively by external standard method by HPLC-UV. The contents of Hederacolchiside A1 and Eleutheroside K in total saponins were (43.0 ± 0.51) % and (5.62 ± 0.03) %, respectively. 2.5. LC-MS/MS analytical conditions All separations were performed on the Agilent 1290 Infinity Rapid Resolution Liquid Chromatography System equipped with a vacuum degasser, a binary pump and an autosampler. Chromatographic separation was achieved on an Agela C18 columnn (4.6×150 mm, 5 m) (Bonna-Agela Technologies Inc.). The column temperature was maintained at 30C, and the injection volume was 10 μL. The temperature of autosampler was at 4C. A gradient mobile phase consisting of solvent A, acetonitrile, and solvent B, 5 mmol/L ammonium acetate was delivered at 1 mL/min and the split ratio was 1:1. Elution conditions were as follows: 0-2.8 min, 60% A; 2.8-2.9 min, 60%-80% A; 2.9-3.5 min, 80% A; 3.5-3.6 min, 80%-60% A; 3.6-5 min, 60% A. The total run time was 5 min. MS analysis was performed on an API 4000 triple quadrupole mass spectrometer equipped with Turbo ion spray sources (TIS sources). The TIS source parameters were as follows: ion spray (IS) voltage: -4000 V; temperature (TEM): 600C; Gas 1 and Gas 2 were set at 50 and 60 psi, respectively. The curtain gas and collision gas were set at 15 and 6 psi, respectively. Quantitative analysis was performed using the multiple-reaction monitoring (MRM) mode. The precursor-to-production pair and compound-dependent parameters, viz. declustering
potential (DP) and collision energy (CE) were optimized for each analyte and selected values are listed in supplementary data. The dwell time for each MRM transition was automatically set at 80 ms by the software. All instrumentation was controlled and synchronized by Analyst software (versions 1.6.) from Applied Biosystems/MDS Sciex. 2.6. Method validation of LC-MS/MS 2.6.1. Preparation of standard solution and quality control samples Standard stock solutions were prepared by dissolving two accurately weighed standards in methanol, two standards (Hederacolchiside A1and Eleutheroside K) were at the same concentration of 500 μmol/mL. A series of working solutions of two analytes were prepared by diluting the mixed standard solutions with methanol in appropriate ratios to yield concentrations of 5-1000 nM for calibration curves and quality control (QC) samples. A solution for internal standard (2 μg/mL) was prepared in methanol. All solutions were stored at -4C. Calibration curves were prepared by spiking with 10 µL different concentrations of the standard working stock solutions to 50 µL blank rat plasma sample subsequently. Calibration curves were prepared at concentrations of 5, 10, 50, 100, 250, 500, 1000 nM for plasma. The similar method was also used to prepare QC samples (namely LLOQ, low QC, med QC and high QC) in rat plasma at concentrations of 5, 20, 200 and 800 nM. 2.6.2. Preparation of plasma samples
An aliquots of 50 μL of rat plasma were spiked with 10 μL of IS solution (2 g/mL) and then precipitate agent of 100 μL acetonitrile was added. The sample had been vortex for 50 s, and centrifuged at 13,000 rpm for 5 min. The supernatant was evaporated to dryness under N2 for about 20 min at 45C. The residues were reconstituted in 100 μL and were centrifuged at 15,000 rpm for 10 min. An aliquot of 10 μL of the final testing samples was injected onto the LC-MS/MS system for analysis. 2.6.3. Method validation The method was validated in terms of specificity, selectivity, calibration curve, sensitivity, matrix effect, accuracy, precision and stability, in accordance with the USA Food and Drug Administration (FDA) bioanalytical method validation guidance [26]. The calibration was performed by least-squares linear regression of the ratio of peak area of each analyte to the IS vs the nominal standard concentration with a weighted factor (1/x). The LLOQ was defined as a reproducible lowest possible concentration, linear with the calibration curves having a relative error between -20% and 20%. To assess the intra-day accuracy and precision of the method, three concentrations levels of QC solution were spiked into plasma, with 6 replicates at each concentration. The extraction recoveries were determined by comparing the peak area obtained from plasma samples spiked before extraction with those from plasma samples spiked after extraction. The matrix effects were evaluated by comparing the peak areas obtained from samples where the extracted matrix was spiked with standard solutions to those obtained from the pure reference standard solutions at the
same concentration. Stability of QC samples was assessed by analyzing samples stored at -70C for 1 month, subjected to three freeze thaw (room temperature) cycles, and stored at room temperature for 24 h. Samples were considered to be stable if the deviation from the nominal concentration was within ± 15%. 2.7. Pharmacokinetic studies of active triterpenoid saponins and the TSS extract On account of the poor solubility in water, Hederacolchiside A1, Eleutheroside K and the TSS extract were suspended separately in an aqueous solution of 0.5% sodium carboxymethyl cellulose (CMC-Na) to get an evenly dispersed suspension for oral administration. For intravenous group, Hederacolchiside A1 and Eleutheroside K were dissolved in (1% DMSO) 0.9% injectable NaCl solution respectively. Eighteen rats were randomly assigned to 6 groups (three rats per group) to be orally administration. In the result: rats in two groups were orally administrated with Hederacolchiside A1 at two dosages of 20 μmol/kg (17.93 mg/kg)and 200 μmol/kg (179.3 mg/kg); two groups were with Eleutheroside K at oral doses of 2 μmol/kg (1.469 mg/kg) and 20 μmol/kg (14.69 mg/kg). The last two groups were oral administrated with the TSS at two oral doses (41.7 and 417.0 mg/kg). It should be noted that the oral dosage (41.7 mg/kg) of the TSS to rats was calculated from the effective dose of Hederacolchiside A1 (20 μmol/kg) while the other oral dosage (417.0 mg/kg) was based on the effective dose of Eleutheroside K (20 μmol/kg). Blood samples (150 μL) were collected from the oculi chorioideae vein of each rat subsequently at 0, 0.33, 0.67, 1, 1.5, 2, 4, 8, 12, 24, 36, 48 h after oral administration.
The collected blood samples were placed in heparinized tubes and separated by centrifugation at 4000 rpm for 5 min. The supernatant was transferred into 1.5 mL eppendorf tubes and stored at -70C prior to analysis. To obtained the value of absolute bioavailability and more pharmacokinetic properties of two makers in rats. Another 2 groups of rats (three rats per group) were administered
an
intravenous
dose
at
0.5
μmol/kg
of
two
components
(Hederacolchiside A1 and Eleutheroside K) separately. Blood samples (150 μL) were collected from the oculi chorioidea vein of each rat subsequently at 0, 0.083, 0.17, 0.5, 1, 2，4, 6, 8, 12, 24, 36 h after intravenous administration. The collected blood samples were conducted in the same manner as above. The animal experiment procedure was approved by the Animal Ethics Board of Shenyang Pharmaceutical University. 2.8. Transport experiments in the Caco-2 cell culture model The Caco-2 cells were used at passages 20-30. The transcellular transport study was performed as described previously [27]. DMSO in DMEM (Dulbecco’s modified Eagle’s medium, 0.1% (v/v), Gibco Invitrogen, Carlsbad, CA) was used as control. After the cells in the flask grew to 90-100% confluency, cells were trypsinized and seeded on collagen-coated polyte-trafluroethylene membrane inserts (0.45 μm) fitted in bicameral chambers (Transwell-COL, 24 mm ID, Corning Inc., Corning, NY) at 1.2 × 105 cells/cm2. The transepithelial electrical resistance (TEER) was tested by Millicell ERS meter (Fisher Sci., Pittsburgh, PA) to reflect the tightness of intercellular junctions and only cells with TEER ≥ 250 Ωcm2 were used for permeability study. Hederacolchiside A1 and Eleutheroside K solution at was loaded
onto the apical or basolateral (donor) side. Three donor samples (500 μL) and three receiver samples (500 μL) were taken at 0, 1, 2, 3, and 4 h followed by the addition of 500 µL of fresh donor solution to the donor side or 500 µL of fresh buffer to the receiver side. The donor side concentrations of the test compounds were always10 µmol/L ether from apical to basolateral or basolateral to apical directions. In the present study, inhibitor (phloridzin 20 µg/mL) of uptake transporter was only added to the apical side of cell monolayers, regardless of where test compounds were loaded. The samples were then analyzed by LC-MS/MS.
2.9. Statistical methods Experimental data and pharmacokinetic parameters are expressed as means ± SD. Pharmacokinetic parameters were calculated by the PK analysis software DAS 2.0 (Chinese Pharmacological Society) using the non-compartmental method. The Cmax and Tmax were observed values without interpolation. The area under the concentration-time curve up to the last measured time point (AUC0-t) was calculated using the trapezoidal rule. The value of AUC0- ∞ was generated by extrapolating AUC0-t to infinity using the elimination rate constant (ke) and the last measured concentration. Apparent elimination half-life (T1/2) was calculated as T1/2 = 0.693/ke, total body clearance (CL) as dose/ AUC0-∞, and apparent volume of distribution (V) as CL/ke. To compare parameters between herb extract group and monomer group, statistical significances were evaluated by Student’s t-tests with computer software SPSS.P value < 0.05 was considered statistically significant. The apparent permeability coefficient (P) was determined by the Equation P = (dQ/dt) A × C0 where dQ/dt is the drug permeation rate (mol/s), A is the surface area of the
epithelium (cm2), and C0 is the initial concentration in the donor compartment at time 0 (nM). Both permeability from the apical to basolateral side (Pab) and basolateral to apical side (Pba) were calculated according to the above equation. The uptake ratio was calculated as Pab/Pba, which represented the degrees of uptake transporter involvement.
3. Results and discussion 3.1. Method development of LC-MS/MS To investigate the pharmacokinetic profiles, it was necessary to develop a rapid, sensitive and robust method for the determination of two triterpenoid saponins in rat plasma. An LC method coupled with tandem mass spectroscopy was developed to achieve this goal. The chromatographic conditions, especially analytical columns and mobile phase compositions (concentration of buffer and percentage of the organic modifiers), were optimized through trials and errors to obtain symmetric peak shape and short run time for the simultaneous analysis of the two compounds. It was observed that acetonitrile gave a better peak shape than methanol, and therefore was selected as the organic phase in mobile phase. Compared with water, the addition of ammonium acetate as a component of the mobile phase was found to make the peak shaper, improve sensitivity and shorten retention time of each analyte. Under the developed chromatographic conditions for simultaneous determination of the two compounds, all analytes were eluted rapidly within 4.0 min. Recent studies have reported the use of ESI-MS for determination of triterpenoid saponins with higher sensitivity and better reproducibility than the other types of ionization [28-30]. In the investigation, both positive and negative modes of
ionization were tested. The response and sensitivity observed in the negative ion mode were higher than that in positive ionization mode. In negative ionization mode, the analytes formed deprotein ions [M−H]−; however, IS produced adduct ion [M+CH3COO]− with acetic acid. Generally, the MS2 fragmentation pathways of saponins were similar involving the loss of various sugar moieties without any parent-ring cleavage which are shown in Fig. 1
3.2. Method validation of LC-MS/MS The chromatogram of a blank plasma sample showed no endogenous peaks interference the analysis of Hederacolchiside A1 and Eleutheroside K. Due to the efficient sample treatment and high selectivity of MRM, matrix effect for these analytes was insignificant. Typical chromatograms of blanks, spiked plasma, along with rat plasma sample after i.g. and i.v. administration are shown in Fig. 2. The calibration curve of Hederacolchiside A1 was y = 0.000208x + 0.00043 (r=0.9969) and y = 0.000898x + 0.00106 (r=0.9995) was for Eleutheroside K. The LLOQs for Hederacolchiside A1 and Eleutheroside K were 5 nM, with accuracy and precision no higher than ± 20% RE and 20% RSD. Table 1 summarized the intra- and the inter-day precision and accuracy of the method. The intra-day and inter-day precisions ranged from 0.73% to 8.06% and 0.73% to 7.88% for both analytes, respectively. The accuracy derived from QC samples was in the range from -3.16% to 3.34%. The result indicated that the assay had good reproducibility with acceptable accuracy and precision.
As shown in Table 2, the mean extraction recoveries of all analytes at different concentrations were from 76.0 to 87.8%. The extraction recovery of IS was 91.0 ± 6.9%. The matrix effect of blank plasma of the analytes and IS was found to be within the acceptable range, suggesting that the plasma matrix effect was negligible for the assay. Stability of the analytes during the sample storing and processing procedures was fully evaluated by analysis of corresponding QC samples. Results of the stability tests shown in Table 3 showed that both of the analytes were stable in plasma samples for 1 month at -70C, 24 h at room temperature (4C) and three freeze-thaw cycles.
3.3. Pharmacokinetic behavior comparison between the consumptions of the TSS versus pure monomers in rats
The plasma concentrations of Hederacolchiside A1 and Eleutheroside K were determined by the established LC-MS/MS method after rats were orally given the TSS extract and the pure monomers at two doses. The mean plasma concentration-time curves of Hederacolchiside A1 and Eleutheroside K after oral dosage are presented in Fig. 3, respectively. The concentration-time curves of both Hederacolchiside A1 and Eleutheroside K had two- or three-peak after oral administration, which might be caused by enteric circulation. This phenomenon is usually observed in the pharmacokinetic study of herbal medicines [31, 32].
The major pharmacokinetic parameters of Hederacolchiside A1 after oral administration of TSS and pure compound calculated by DAS 2.0 software are listed in Table 4. After oral administration of TSS, Hederacolchiside A1 reached the maximum concentration (Cmax) of (41.77 ± 10.90) nmol/L and (80.00 ± 14.64) nmol/L at the doses of 41.7 and 417.0 mg/kg, respectively. The area under curve of the concentration-time profile (AUC0-t) were (209.85 ± 26.08) nmol·h/L at the dose of
41.7 mg/kg and (694.17 ± 82.03) nmol·h/L following the dose of 417.0 mg/kg. However, the values of Cmax and AUC0-t increased significantly after the equivalent oral doses of Hederacolchiside A1 monomer. The Cmax value of Hederacolchiside A1 after dosage of monomer at 20 μmol/kg (17.93 mg/kg) and 200 μmol/kg (179.3 mg/kg) was (90.69 ± 26.96) nmol/L and (1069 ± 304.77) nmol/L, respectively. The correspondent AUC0-t of Hederacolchiside A1 were (378.21 ± 58.89) nmol·h/L and (112104.96 ± 1727.08) nmol·h/L, respectively. The time of peak concentration (Tmax), and half-life (T1/2) of Hederacolchiside A1 after oral administration of TSS and pure compound did not show significant differences.
Similar to Hederacolchiside A1, the Cmax and AUC0-t data of Eleutheroside K after TSS (417.0 mg/kg) administration were statistically lower than those after administration of equivalent dose of pure monomer 20 μmol/kg (14.69 mg/kg). When rats were orally treated with TSS at 417.0 mg/kg, the Cmax of Eleutheroside K was (15.45 ± 5.59) nmol/L and the AUC0-t was (69.40 ± 9.39) nmol·h/L. As shown in Table 5, when rats were given an oral dose of 20 μmol/kg (14.69 mg/kg) pure monomer, the Cmax and AUC0-t values of Eleutheroside K were (48.13 ± 14.87) nmol/L and (136.89 ± 37.67) nmol·h/L. However, after rats were orally admistrated with lower dosage of TSS at 41.7 mg/kg or Eleutheroside K monomer at 2 μmol/kg (1.469 mg/kg), there were no significant differences in the parameters between the two groups.
The results indicated that the pure compounds were more efficient than the TSS extract in the absorption of Hederacolchiside A1 and Eleutheroside K. The present
study was the first to report the difference existing in pharmacokinetic profiles of the test active compounds administrated in herb extract and monomer. Zeng et al. reported that the oral bioavailability of pure baicalin was higher than that of the herbal preparation of Huang-Lian-Jie-Du-Tang, a formulation containing Rhizoma Coptidis, Radix Scutellariae, Cortex Phellodendri and Fructus Gardenia [33]. They illuminated that the lower oral bioavailability of baicalin after administration of herbal preparation was resulted from the inhibited absorption of baicalin by berberine, a quaternary ammonium compound [34]. The mechanisms of the worse absorption of Hederacolchiside A1 and Eleutheroside K following dose of TSS were not investigated in the present study. We speculated that some ingredients in TSS extract weaken the absorption of both active ingredients. Further studies are scheduled to elucidate the underlying mechanism of the pharmacokinetic differences.
In the present study, we also conducted the i.v. pharmacokinetics of pure Hederacolchiside A1 and Elutheroside K to study the absolutely bioavailability. The concentration-time curves of Hederacolchiside A1 and Eleutheroside K after i.v. administration are shown in Fig. 3. Elutheroside K exhibited rapid elimination while Hederacolchiside A1was moderate after i.v. injection, which was also observed after oral administration. In the previous literatures, most of saponins were susceptible to rapid and extensive biliary excretion through active transport, which may lead to short T1/2, low systemic exposure [35]. It’s valuable that Hederacolchiside A1 such a long-circulating saponin used as pharmacokinetic marker to substantiate systemic exposure to the ingested herb extract. Furthermore, the absolute bioavailability of
pure Elutheroside K was 0.30% and the value of Hederacolchiside A1 was 0.141% at the same oral dosage. The low oral bioavailability of the two triterpenoid saponins might be attributed to their poor gastrointestinal absorption [36,37], because their molecular masses (733.5 Da and 896.5 Da) were larger than the favorable value (<500 Da) for membrane permeability [38]. Further studies were performed to investigate the absorption mechanism of the two pure compounds.
3.4. Absorption mechanism in the Caco-2 cell The absorption permeability of Hederacolchiside A1 (3.09 ± 0.33×10-6 cm/sec) was significantly higher than that of efflux permeability (0.61 ± 0.20×10 -6 cm/sec). Notably, the uptake ratio of 5.03 indicated that uptake transporter(s) was involved in the absorption of Hederacolchiside A1. However, the uptake ratio was declined to 2.03 when phloridzin, a SGLT1 inhibitor, was added into the apical side, suggesting that SGLT1 probably participate in the active absorption of Hederacolchiside A1. The transporter behavior of Eleutheroside K in the Caco-2 cell culture model was tested in a similar method to that of Hederacolchiside A1. When (SGLT1 inhibitor) phloridzin exist, the uptake permeability decreased significantly (from 2.87 ± 0.51×10-06 cm/sec to 1.76 ± 0.31×10-06 cm/sec) while the efflux permeability was (from 1.52 ± 0.45×10-6 cm/sec to 1.26 ± 0.78×10-6 cm/sec) (Fig. 4). SGLT1 also influenced the transport of Eleutheroside K.
The Caco-2 cell monolayer system was a standard convenient model to study human intestinal absorption of xenobiotic compounds [39]. The relatively low permeability values of about 10-6 cm/sec for Hederacolchiside A1 and Eleutheroside K illuminated their weak permeation across the intestinal tract, which was agreed with their poor
oral bioavailability. In addition, the transporter proteins involved in the active influx of both saponins were identified. The transporter SGLT1 present in the apical membrane of the small intestine seemed participating in their active absorption [40, 41]. The pharmacokinetic (PK) and absorption studies of the two active compounds discussed herein will be more productive for the exploration of TSS’s pharmacological activity.
Conclusion
A reliable LC-MS/MS method was developed for the simultaneous determination of Hederacolchiside A1and Eleutheroside K in rat plasma. It was successfully applied to the comparative pharmacokinetic studies between consumptions of the TSS versus pure monomers in rats. The result of the present study demonstrated that both Hederacolchiside A1 and Eleutheroside K showed poor absorption after oral administration and TSS matrix inhibited their absorption. In addition, Transport experiments in the Caco-2 cell culture model showed low permeability (Pab) of Hederacolchiside A1 and Eleutheroside K. Absorption of Hederacolchiside A1 was mainly through positive transport and SGLT1 might participate in its active absorption.
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A
notification
on
the
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of
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Fig.1. Product ion spectra and monitored transitions of analytes.
Fig.2. Representative extract ion MRM chromatograms of two marker compounds and Triamcinolone acetonide (IS): (A) blank plasma, (B) blank plasma spiked with the two analytes at LLOQ and IS, (C) 2 h sample plasma after a single oral administration of the TSS extract at 417.39 mg/kg.
B
A
C D
.
E
F
Fig.3. Mean plasma concentration vs time profile of Hederacolchiside A1 and Eleutheroside K: (A) after oral administration of TSS extract (41.7 and 417.0 mg/kg), (B) after oral administration of Hederacolchiside A1 (20 μmol/kg and 200 μmol/kg), (C) after oral administration of TSS extract (41.7 and 417.0 mg/kg), (D) after oral administration of Eleutheroside K (2 μmol/kg and 20 μmol/kg), (mean ± SD, n = 3), (E)after intravenous administration of
Hederacolchiside A1 to rats (0. 5 μmol/kg), (F)after
intravenous administration of Eleutheroside K to rats (0. 5 μmol/kg) (mean ± SD, n = 3).
Fig.4. Transport of Hederacolchiside A1and Eleutheroside K in the Caco-2 cell culture model. The donor side concentrations of the test compounds were always 10 µmol/L either from apical to basolateral or basolateral to apical directions while phloridzin (20 µg/mL) in inhibitor group. Each data point is the average of three determinations. Bars are standard deviations of the mean. The asterisk (*) indicates P < 0.05 when A→B group compared with the corresponding B→A group of compounds.
Table 1 Precision and accuracy of two marker compounds for LC-MS/MS method (n = 6). Spiked Components (nM) Hederacolchiside A1
Eleutheroside K
Concentration
Intra-day
Inter-day
Measured
Precision
Precision
(nM)
(RSD, %)
(RSD, %)
Accuracy
(RE, %)
5
4.96 ± 0.22
1.11
1.25
-0.83
20
19.67 ± 1.10
5.24
5.27
-1.65
200
194.06 ± 11.30
6.19
0.92
-2.90
800
789.62 ± 61.40
8.06
1.72
-1.30
5
4.96 ± 0.15
0.73
0.73
-0.85
20
20.67 ± 0.99
4.24
7.88
3.34
200
197.63 ± 11.83
5.74
7.60
-1.18
800
774.69 ± 35.97
4.74
2.41
-3.16
Table 2 Recoveries and matrix effects of two marker compounds for LC-MS/MS method (n = 6). Components
Spiked (nM)
Extraction recovery (%)
Matrix effect (%)
Hederacolchiside A1
20
87.8 ± 11.5
102.7 ± 11.7
200
76.0 ± 5.6
101.4 ± 7.9
800
76.1 ± 8.0
99.5 ± 7.7
20
77.2 ± 9.1
100.4 ± 12.3
200
78.2 ± 3.1
100.6 ± 8.1
800
76.2 ± 3.4
95.7 ± 12.2
91.0 ± 6.9
101.7 ± 7.2
Eleutheroside K
IS
Table 3 Stability for analysis of two marker compounds in rat plasma (n = 6) Spiked
Short term (25°C, 24 h)
Long term (-25°C, 30 days)
Freeze-thaw
(nM)
RSD (%)
RE (%)
RSD (%)
RE (%)
RSD (%)
RE (%)
20
2.7
-2.85
2.78
-1.03
3.8
-1.33
800
4.43
7.49
4.1
7.71
4.49
1.625
20
5.18
-0.67
4.5
0.17
4.35
9.17
800
1.66
-5.88
3.84
-8
3.86
-4.46
Components
Hederacolchiside A1
Eleutheroside K
Table 4 Pharmacokinetic parameters of Hederacolchiside A1 after single oral administration of TSS extract at doses of 41.7 and 417.0 mg/kg, intraveneous administration of pure monomer at 0.5 μmol/kg and single oral administration of pure monomer at 20 and 200 μmol/kg to male rats (mean ± SD).
Component
Oral
Intraveneous
Oral
TSS
Monomer
Monomer
41.7 mg/kg
417.0 mg/kg
0.5 μmol/kg
Cmax (nmol/L)
41.77 ± 10.90
80.00 ± 14.64
AUC0-t (nmol·h/L )
209.85 ± 26.08
694.17 ± 82.03
Tmax (h)
3.33 ± 1.15
2.78 ± 2.12
T1/2 (h)
6.68 ± 6.84
15.86 ± 4.54
AUC0-∞ (nmol ·h/L)
263.93 ± 32.12
819.85 ± 141.61
20
μmol/kg
200
μmol/kg
(17.93 mg/kg)
(179.3 mg/kg)
9331.33 ± 2025.51
90.69 ± 26.96*
1069.00 ± 304.77**
12059.48 ± 3741.98
378.21 ± 58.89*
112104.96 ± 1727.08**
3.17 ± 2.46
10.00 ± 3.46
8.80 ± 1.31
5.96 ± 2.93
6.90 ± 2.44
12274.76 ± 3763.37
688.42 ± 330.59
12264.77 ± 1742.96
0.141 ± 0.068
0.251 ± 0.036
Hederacolchiside A1
V (L/Kg)
0.023 ± 0.010
CL (L/h/Kg)
0.002 ± 0.001
a
F (%)
0.054 ± 0.007
0.017 ± 0.003
The oral administration of TSS extract at 41.7 and 417.0 mg/kg was equivalent to the oral administration of Hederacolchiside A1at 20 μmol/kg and 200 μmol/kg. **P < 0.01 when compared with the corresponding group of TSS extract, *P < 0.05 when compared with the corresponding group of TSS extract. a
F (%) = [(Doseiv × AUC0–∞oral)/(Doseoral × AUC0–∞iv)] × 100
Table 5 Pharmacokinetic parameters of Eleutheroside K after single oral administration of TSS extract at doses of 41.7 and 417.0 mg/kg, intraveneous administration of pure monomer at 0.5 μmol/kg and single oral administration of pure monomer at 2 and 20 μmol/kg to male rats (mean ± SD).
Component
Oral
Intraveneous
Oral
TSS
Monomer
Monomer 2 μmol/kg
41.7 mg/kg
417.0 mg/kg
0.5 μmol/kg (1.469mg/kg)
20
μmol/kg
(14.69 mg/kg)
Cmax (nmol/L)
12.25± 4.34
15.45 ± 5.59
5233.33 ± 841.27
12.70 ± 2.19
48.13 ± 14.87*
AUC0-t (nmol ·h/L)
53.82 ±28.03
69.40 ± 9.39
1597.39 ± 90.89
40.21 ± 12.15
136.89 ± 37.67*
Tmax (h)
2.75 ± 1.77
5.00 ± 3.61
4.78 ± 3.98
1.50 ± 0.50
T1/2 (h)
4.51 ± 4.11
8.94 ± 1.71
0.8 0 ± 0.40
3.93 ± 2.84
1.74 ± 0.57
AUC0-∞ (nmol·h/L)
81.77 ± 9.38
196.85 ± 16.42
1601.77 ± 84.39
97.03 ± 3.41
191.35 ± 95.32
1.521 ± 0.053
0.300 ± 0.149
Eleutheroside K
V (L/Kg)
0.014 ± 0.008
CL (L/h/Kg)
0.012 ± 0.001
a
F (%)
1.282 ± 0.147
0.309 ± 0.026
The oral administration of TSS extract at 41.7 and 417.0 mg/kg was equivalent to the oral administration of Eleutheroside K at 2 μmol/kg and 20 μmol/kg. *P < 0.05 when compared with the corresponding group of TSS extract. a
F (%) = [(Doseiv × AUC0–∞oral)/(Doseoral × AUC0–∞iv)] × 100