Article pubs.acs.org/JAFC
Gastrointestinal Absorption and Metabolic Dynamics of Jujuboside A, A Saponin Derived from the Seed of Ziziphus jujuba Panpan Song,† Yan Zhang,† Guijie Ma,† Yanqing Zhang,*,†,‡ Aimin Zhou,†,§ and Junbo Xie*,†,‡ †
College of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin, 300134, China Tianjin Key Laboratory of Food Biotechnology, Tianjin 300134, China § Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115, United States ‡
S Supporting Information *
ABSTRACT: Jujuboside A (JuA), an active saponin, is responsible for the anxiolytic and sedative effects of Zizyphi Spinosae Semen (ZSS). In this study, the gastrointestinal absorption and metabolic dynamics of JuA were investigated in vivo and in vitro. The results showed that the bioavailability was 1.32% in rats, indicating only a trace amount of JuA was able to be absorbed. Further investigation revealed that its poor bioavailability was not caused by malabsorption but by the metabolic process. JuA was hydrolyzed largely in the stomach before being absorbed into the different parts of the intestine (especially duodenum and colon), and the gastric environment played a vital role in this process. Furthermore, the metabolites, jujuboside B (JuB) and jujubogenin, exhibited significant effects on the expression and activation of γ-amino-butyric acid A (GABA(A)) receptors. Our findings demonstrate that the metabolites of the saponin, not the original molecule, should be responsible for the specific bioactivities. KEYWORDS: bioavailability, intestinal absorption, Jujuboside A, metabolic dynamics, Zizyphi Spinosae Semen
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INTRODUCTION Zizyphi Spinosae Semen (ZSS), the dried seeds of Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou, is one of the most popular traditional Chinese herbal foods for the treatment of fright palpitations, insomnia, and dreaminess.1 It has been reported that ZSS displays anxiolytic,2 hypnotic,3,4 memory modulating,5 and cardiotonic activities.6 Saponins are believed to be the main constituents of ZSS responsible for its anxiolytic and sedative effects. Studies have demonstrated that JuA is one key saponin in ZSS, contributing to its sedativehypnotic effect.7−9 JuA has an inhibitory effect on the rat hippocampal formation in vivo and in vitro and decreases the slopes of excitatory postsynaptic potential through the glutamate mediated excitatory signal pathway.10,11 It can also modulate the expression of the GABA receptor subunit genes in hippocampal neurons.12,13 Recently, there have been many reports suggesting that not JuA but its metabolite (jujubogenin) is really responsible for the sedative bioactivity through interacting with GABA(A) receptors.14,15 However, the conclusion was drawn from virtual screening and molecular dynamic simulation and on the premise that JuA cannot be absorbed due to its large molecular volume. In recent years, it has been demonstrated that JuA is able to be found in the blood of rats after oral administration.16 Nevertheless, the intestinal absorption characteristics and bioavailability of JuA are still vague up to now. It is well-known that there are many factors affecting the bioavailability of food or drugs. As a specific food component, the level of its absorption in the gastrointestinal tract is the most important criteria for interpreting its bioavailability.17 In addition, its metabolism in the gastrointestinal tract is also affected by a variety of factors, such as gastric acidity, gut © 2017 American Chemical Society
microbiota, intestinal membrane enzymes, complexion with food constituents, bacterial enzymes, and so on.18 No doubt, these factors are able to directly impact the bioavailability of the component through the metabolic process to change its prototype.19 Indeed, studies have revealed that the effective molecules of functional food are usually not resulted from the original constituents themselves but the degraded products in the gastrointestinal tracts.20,21 Thus, how the components are metabolized in the gastrointestinal tract has become an attractive subject in studying a functional food.22 Our previous report has shown that JuA can be decomposed significantly as being incubated with rat feces in vitro.23 However, how JuA is metabolized in the gastrointestinal tract remains to be fully understood. In the present study, the bioavailability of JuA was evaluated through area under the plasma concentration−time curve (AUC) in vivo. The absorption of JuA in the different intestinal segments of the rats was examined to confirm the bioaccessibility of the saponin. The metabolic dynamics of JuA in the gastrointestinal contents and the submucosal organelle environment was determined to interpret its bioavailability. Furthermore, the main metabolites of JuA were identified with mass spectrometry (MS). The effects of its main degradation metabolites on mRNA expression and activation of GABA(A) receptors in cultured rat hippocampal neurons were determined by real time fluorescence quantitative reverse transcription polymerase chain reaction (RTFQ-PCR) Received: Revised: Accepted: Published: 8331
June 14, 2017 August 28, 2017 September 4, 2017 September 4, 2017 DOI: 10.1021/acs.jafc.7b02748 J. Agric. Food Chem. 2017, 65, 8331−8339
Article
Journal of Agricultural and Food Chemistry
In the intravenous administration group, the rats were injected through tail vein with 4 mg·kg−1 of JuA (dissolved in physiological saline solution), while the rats were administrated with 25 mg·kg−1 JuA by gavage for the other group. Blood samples (0.3 mL) were drawn from the fundus vein at 10 min, 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h after dosing. The plasma sample (100 μL) was treated according to the method described in Preparation of Standard Solutions and Calibration Curve. Calculation of Bioavailability and Other Pharmacokinetics Parameters. Pharmacokinetics parameter calculations were performed using the Noncompartmental model.24 The maximum plasma concentration (Cmax) and the time to reach Cmax (Tmax) were directly obtained from the experimental data. The elimination half-life (t1/2) was calculated as 0.693/Ke, where Ke is the elimination rate constant calculated from the terminal linear portion of the log plasma concentration−time curve. The area under the plasma concentration−time curve from time zero to the last quantifiable time point (AUC0→10) and from time zero to infinity (AUC0→∞) were estimated using the log−linear trapezoidal rule. Bioavailability (F) can be interpreted as the degree of absorption of a dietary component or drug. It can be calculated by comparing the values of AUC after intravenous and oral administration. The above comparison is feasible for the same individual assuming that the Ke and Vd values are constant and the component can be used entirely (100%) after intravenous administration. The formula is
and the N-ethoxycarbonylmethyl-6-methoxyquinolinium bromide (MQAE) fluorescence probe technique, respectively. Our findings would reveal the bioactivity mechanism of the effective component in ZSS.
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MATERIALS AND METHODS
Chemicals and Reagents. JuA (purity over 99%) were purified and further characterized by ultraviolet spectra, infrared spectroscopy, MS, and nuclear magnetic resonance in our laboratory. The standard substance of JuA (PubChem CID: 51346169), JuB, notoginsenoside R1 and jujubogenin were purchased from Shanghai Tongtian Biotechnology Co., Ltd. Diazepam came from Tianjin Jinyao Amino Acid Corporation (Tianjin, China). Pepsin and trypsin were bought from Beijing Dingguo Biotechnology Co. Phenol red was obtained from Tianjin Chemical Reagent factory. Chromatographic pure methanol was from Merker (Darmstadt, Germany). Chromatographic pure water was purchased from J. T. Baker (J. T. Baker Chemicals, USA). Fetal bovine serum, penicillin-streptomycin, DMEM/F12, Neurobasal-A (without phenol red) and B27 culture medium supplement were all purchased from Gibco (USA). All other reagents used in the experiment were commercially available and were of analytical grade. Animals. Sprague−Dawley (SD) rats (200 ± 20 g, 6−8 weeks) were obtained from Tianjin Institute of Materia Medica (Tianjin, China). All the experimental procedures were demonstrated to be ethically acceptable and conformed to the National Guidelines on the Proper Care and Use of Animals in Laboratory Research (2011 version). The permission number of using these rats was TCMLAEC2015027. Instrumentation and HPLC-MS/MS Conditions. The Agilent 1100 series high performance liquid chromatography system consisted of a G1312B pump, a G1316B column temperature compartment, and a G1367D automatic sampling device (Agilent, USA). The separation was performed with an YMCTM ODS-AQ S-5 (2.0 × 100 mm, 3 μm) column using a mobile phase consisting of methanol and 0.1% formic acid (50/50, v/v) at 30 °C. The flow rate was 0.3 mL·min−1. A G6410B Triple Quad LC/MS (Agilent, USA), interfaced via an electrospray ionization (ESI) probe, was operated in the negative ion mode with multiple reaction monitoring (MRM). The characteristic transitions of precursor [M − H]− to product ions were m/z 1205.1 → 749.5 (quantifier), 1205.1 → 1073.2 (qualifier) for JuA and 931.7 → 799.6 for notoginsenoside R1 (internal standard, IS) respectively. The optimized conditions were as follows: heater temperature (350 °C), ion source voltage (4000 V), curtain gas flow rate (N2, 8.0 L·min−1), nebulizer gas pressure (N2, 35 p.s.i), fragmentor (JuA: 360 V; notoginsenoside R1: 105 V), collision energy (JuA: 60 eV; notoginsenoside R1: 47 eV). Bioavailability Investigation of JuA in Vivo. Preparation of Standard Solutions and Calibration Curve. The standard solution of 1 mg·mL−1 JuA was diluted with methanol into a liner concentration gradient: 4, 10, 40, 80, 200, 400, 800, 1000 ng·mL−1. 100 μL of each concentration solution and 50 ng·mL−1 notoginsenoside R1 were charged in Eppendorf tubes respectively and dried with N2. After the residue was mixed with 100 μL blank plasma, 200 μL methanol was added into the tubes, vortexed for 5 min, and centrifuged at 6000 g for 10 min. The upper organic phase was carefully transferred to another tube, and dried with N2. Then 100 μL 50% methanol was added to dissolve the residue. After being filtered with 0.45 μm membrane, 20 μL of the sample solution was injected into the HPLC−MS/MS system. The calibration curve was obtained by plotting the peak area ratios of JuA to the IS versus the concentrations of JuA and assessed by weighted least-squares linear regression using 1/x2 as weighting factor. Collection and Processing of the Sample. Twelve SD rats were evenly divide to two groups (intravenous administration group and oral administration group), and were kept on a 12 h light/dark cycle with free access to standard diet and water. After being acclimated for 7 days, the rats were fasted for 24 h and had free access to water before dosing.
F (%) =
Div × AUCig Dig × AUCiv
× 100% (1)
,where D is the dose. Intestinal Absorption Kinetics Investigation in Rats. Chromatography and Mass Spectrometry Conditions. The optimized mass spectrometric conditions of JuA were the same as above. The transition of phenol red was m/z 353.0 → m/z 195.1. The fragmentor and collision energy were optimized as 180 V and 40 eV respectively. Collection and Processing of the Sample. In situ perfusion of the intestine is a commonly used approach to investigate the intestinal absorption characters of food components.25,26 In this study, the method was used to investigate the intestinal absorption of JuA in rats. The SD rats were fasted for 24 h before the experiment. After fixation and anesthesia, the rats were ligated and the circulation device was installed. To investigate the absorption of JuA in different intestinal parts, the ligation was performed with various modes, including entire intestine, duodenum, jejunum, ileum and colon. Before circulation, the ligated intestinal parts were rinsed fully with normal saline (37 °C). Since water can be absorbed in the various parts of the intestine, phenol red, which cannot penetrate the wall of the intestine under the certain pH conditions, is used as the internal standard to calibrate the changed volume of the reflux liquid. 100 mL Kreb-Ringer’s buffer solution (containing 20 μg·mL−1 phenol red and 20 μg·mL−1 JuA) was circulated at a speed of 5 mL·min−1 for 10 min and then adjusted to be 2.5 mL·min−1 (0 time point). At 0, 0.5, 1, 1.5, 2, 3, 4 h, 1 mL sample was collected respectively, and 1 mL of blank phenol red solution (only containing 20 μg·mL−1 phenol red) prepared by Kreb-Ringer’s buffer was supplemented. The sample was centrifuged and filtered through a 0.45 μm membrane. The concentration was determined by the HPLC-MS/MS method and calculated with the calibration equation: Y = 368.48X + 142.26 (R2 = 0.9978). Calculation of the Absorption Percentage. The absorption percentage (X%) was calculated as follows:
X (%) =
C0V0 − (C0′V0 /C1′) × C1 × 100% C0V0
(2)
,where C0 is the initial concentration of JuA solution, C1 is the determined JuA concentration in the circulating solution, C0’ is the initial phenol red concentration, C1’ is the determined phenol red concentration in the circulating solution, and V0 is the original volume of the sample solution. A straight line was obtained by plotting the 8332
DOI: 10.1021/acs.jafc.7b02748 J. Agric. Food Chem. 2017, 65, 8331−8339
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Journal of Agricultural and Food Chemistry
Effects of the Metabolites on Hippocampal Neurons. Culture and Purity Identification of Hippocampal Neurons. The hippocampal neurons from Sprague−Dawley rats (postnatal 1−3 days) were prepared and cultured referring to the reported method.29 The purity of the neurons was evaluated through immunofluorescence staining.30 The hippocampal neurons were cultured for 8 days and rinsed with ice phosphate buffer solution (PBS). Then, the cells were fixed for 4 min at room temperature with 4% paraformaldehyde. After being washed with PBS, the cells were dealt with 0.3% TritonX-100 to make it transparent. 100 μL of rabbit anti-MAP2 antibody (Abcam, USA) and an Alexa Fluor 488 donkey antirabbit IgG (H+L) antibody (Invitrogen, USA) were added successively. After being added DAPI, fluorescence observation was performed by fluorescence inverted microscope. MTT Assay. Hippocampal neurons (cultured for 3 days) were seeded into 96-well plates and incubated in the incubator. After the cells reached to 70−80%, they were cultured with 100 μL of PBS (blank control) and metabolites for 24 h.31 The absorbance was assayed on the microplate reader at 490 nm.
logarithm of the amount of residual JuA (ln X) in the intestine versus the sampling time t:
ln X = ln X 0 − K at
(3)
,where Ka is the absorption rate constant, X0 is the initial amount of JuA. Determination of the Liver Portal Vein Blood Samples. To further determine the absorption situation of JuA, its concentration in rat liver portal vein blood samples with various ligation modes of intestinal parts was measured by using the method of portal vein catheterization. Metabolic Dynamics in Vitro. Preparation of Gastrointestinal Tract Contents and Mucosal Subcellular Organelles. First of all, the artificial gastric juice and artificial intestinal fluid were prepared using the reported method.27 After being fasted for 48 h with free access to water, three SD rats were anesthetized and their stomach, duodenum, jejunum, ileum and colon were collected. The contents in stomach and various intestinal parts were rinsed and gathered respectively with 10 mL of artificial gastric juice and physiological saline. The mixture was centrifuged (6,000 g) for 15 min and then the supernatant was stored at −70 °C. The mucosa of the stomach and intestine were scraped and suspended in 0.3 M sucrose solution (pH 7.0), and homogenized with ultrasonic method. Subsequently, the homogenate was centrifuged at 10,000g for 1 h at 4 °C, and the supernatant was used as cytosol. While mixing the precipitate with sucrose solution and 1 M HAc/NaAc buffer (pH 4.5) respectively, the brush border membrane (BBM) and lysosome were obtained. Preparation of Standard Curve. The standard curve was obtained by plotting the peak area ratios of JuA to IS versus the concentrations of JuA. After 400 μL of the standard solutions (1, 2, 4, 8, 10, 20 μg· mL−1, containing IS) were dried with N2, 400 μL sucrose solution (0.3 M), Kreb-Ringer’s buffer solution, 1 M HAc/NaAc buffer solution (pH 4.5), HCl solution (pH 1.2), artificial gastric juice and artificial intestinal fluid were mixed with the residue, respectively. The mixture was vortexed for 5 min, thus the standard curve solutions were obtained. The solutions (20 μL) were injected into HPLC-MS/MS after being diluted with mobile phase and filtered. Metabolic Dynamics of JuA in Vitro. The gastric environment was simulated in vitro, and the gastric content was suspended with HCl solution (pH 1.2) to 10 μg·mL−1 (calculated as protein concentration28). Likewise, the intestinal environment was simulated, and the content of each intestinal segment was diluted to 10 μg·mL−1 with Kreb-Ringer’s buffer (pH 7.4). The same concentration of cytosol, brush border and lysosome derived from each gastrointestinal segment was prepared with 1 M HAc/NaAc buffer solution (pH 4.5). Before incubation, all of these incubation solutions were activated for 30 min at the temperature of 37 °C. For the metabolic dynamics investigation of JuA, 400 μL of the standard solution (200 μg·mL−1, containing IS) was put in a glass tube, and evaporated to dryness with N2. The residue was mixed with the above incubation solution (including artificial gastric juice and artificial intestinal fluid) to obtain 20 μg·mL−1 concentration (n = 3), and cultured at 37 °C. 50 μL solution was collected at 0, 1, 2, 3, 4, 6, 8, 10, 12, 24, and 48 h, respectively. The samples were diluted with the mobile phase before being filtered. The content of JuA in the sample was calculated according to the standard curve. Identification of the Metabolites in Vitro. According to the chemical structure of JuA (M0), the glycosyl group in the side chain could be removed step by step during the metabolic process, and the product structure was predicted and the molecular weight could be calculated. The full scan and the product ion scan were used to obtain the m/z of the parent ion and the product ion of the metabolites. The detection conditions to determine the metabolites (derived from incubation with gastric contents) were optimized. Three main metabolites (M1, M2, M3) were identified and deduced. The transitions of M1, M2 and M3 were m/z 1043.5 → 911.2/749.1, m/z 911.5 → 749.2 and m/z 471.2 → 433.2 respectively. The fragmentor and collision energy were optimized as 320 V and 40 eV for M1, 340 V and 30 eV for M2, 90 V and 10 eV for M3. The other chromatographic conditions and mass spectrometry conditions were as shown in the section Instrumentation and HPLC-MS/MS Conditions.
survival rate =
A × 100% A0
(4)
,wherein A was the average absorbance value of treatment group, and A0 was the average absorbance value of blank control group. Effects of Metabolites on GABA(A) Receptor Gene Expression and Receptor Activation. To study the effect of metabolites on GABA(A) receptor expression, RTFQ-PCR was used to determine the mRNA level of GABA(A) receptors in cultured rat hippocampal neurons. The primers were designed with Premier 5.0, and their sequences were shown in Table S1 (Supporting Information). The β-actin was used as the internal control. RTFQ-PCR was performed in 20 μL reaction mixtures containing 2 μL cDNA, 0.6 μM forward and reverse primers, 10 μL FastStart Universal SYBR Green Master (Rox) (Roche, USA). The activation of GABA(A) receptor was evaluated by the change of chloride ion concentration in the hippocampal neurons by using MQAE fluorescence probe technique.32 Hippocampal neurons were cultured with different concentrations of JuB, jujubogenin (0, 25, 50 μg·mL−1) and diazepam (10 μg·mL−1, positive control group) for 24 h. Then, the solution was replaced by 100 μL of 5 mM MQAE incubation solution, and the supernatant was discarded after 1 h of incubation at 37 °C. Finally, the cells were washed with Krebs-HEPES buffer for 3−5 times, and fluorescence values were measured in the microplate reader (excitation wavelength 365 nm, emission wavelength 460 nm). Statistical Analysis. The Agilent MassHunter Workstation Software (Agilent Technologies) and OriginLab Origin 8.1 were performed to analyze the chromatogram and data processing, respectively. The data were presented as the mean ± SD (standard deviation). The results would be considered statistically significant when P value was below 0.05.
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RESULTS Bioavailability of JuA in Vivo. The pharmacokinetics of JuA in rats after intravenous injection and oral administration was analyzed by a HPLC-MS/MS method. The calibration curve exhibited excellent linearity in the range of 4−1000 ng· mL−1. The typical calibration equation was y = 4.75x + 36.35 (r2 = 0.9992). The mean plasma concentration−time profiles of JuA after intravenous injection and oral administration are illustrated in Figure 1, and the pharmacokinetics parameters for these different routes of administration are listed in Table 1. The AUC0→10 and AUC0→∞ values of intravenous injection and oral administration were 2839.89 ± 255.34 ng·mL−1·h, 206.02 ± 13.33 ng·mL−1·h and 3201.51 ± 282.87 ng·mL−1·h, 211.13 ± 11.75 ng·mL−1·h, respectively. The bioavailability of JuA was only 1.32% via oral administration in rats. Moreover, the value of t1/2 (1.35 h-2.55 h) indicated that JuA underwent rapid elimination or strong metabolism in vivo. The typical HPLC− 8333
DOI: 10.1021/acs.jafc.7b02748 J. Agric. Food Chem. 2017, 65, 8331−8339
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Journal of Agricultural and Food Chemistry
Intestinal Absorption Kinetics in Rats. Chromatography and Mass Spectra. The typical HPLC−MS/MS chromatogram of the samples is shown in Figure 2B, indicating that JuA and phenol red had a good separation, which could be used in subsequent experimental studies. Intestinal Absorption. The absorption rate constant (Ka) and absorption percentage of JuA in duodenum, jejunum, ileum, and colon are shown in Table 2. The results showed that Table 2. Absorption Rate Constants (Ka) and Absorption Percentage (X%) of JuA in Different Intestinal Segments (n = 3) Intestinal segments Duodenum Jejunum Ileum Colon
Figure 1. Plasma concentration−time profiles of JuA. C1: Oral administration with 25 mg·kg−1; C2: Intravenous administration with 4 mg·kg−1 (n = 6).
Unit
ke t1/2 Tmax Cmax AUC0−10 AUC0‑∞ MRT0−10 MRT0‑∞
h−1 h h ng·mL−1 ng·mL−1·h ng·mL−1·h h h
Intravenous administration 0.28 ± 0.03 2.55 ± 0.35
2839.89 ± 255.34 3201.51 ± 282.87 2.76 ± 0.12 3.33 ± 0.27
0.0995 0.0031 0.0218 0.0645
± ± ± ±
X (%)
0.0005 0.0007 0.0027 0.0100
41.0082 11.6855 25.5926 33.4004
± ± ± ±
2.9700 1.0831 4.4597 4.2424
the absorption of JuA is distinct in different parts of the intestine and that there is a significant difference between the absorptions in each part (Figure 3A). Obviously, the Ka and the absorption percentage of JuA in the duodenum (41.0082 ± 2.9700) were significantly higher than those in other intestinal parts. In addition, the absorption of JuA in the colon (33.4004 ± 4.2424) was more preferable when compared with other intestinal segments except the duodenum (Figure 3B). Determination of the Liver Portal Vein Blood Samples. After 4 h of circulation in the total intestinal segment (the initial concentration of 20 μg·mL−1), JuA was determined as 2.24 μg·mL−1 in the liver portal vein blood sample. Likewise, the assayed concentration was 1.05 μg·mL−1 after 4 h of circulation in the duodenum and colon, and 0.65 μg·mL−1 after the circulation in the jejunum and ileum. Therefore, the amount of JuA absorbed through the duodenum and colon was higher than that via the jejunum and ileum, demonstrating that the duodenum and colon are the better absorption sites.
Table 1. Pharmacokinetics Parameters of JuA in Rats Following Intravenous Administration (4 mg·kg−1) and Oral Administration (25 mg·kg−1) of JuA (n = 6) Parameters
Ka (h−1)
Oral administration 0.51 ± 0.03 1.35 ± 0.17 2.00 42.84 ± 4.36 206.02 ± 13.33 211.13 ± 11.75 3.79 ± 0.15 3.84 ± 0.16
MS/MS chromatograms of JuA after intravenous and oral administration are shown in Figure 2A.
Figure 2. Representative TIC chromatograms of the plasma samples. (A) Bioavailability investigation study (JuA and Notoginsenoside R1). (B) Intestinal absorption study (JuA and Phenol red). 8334
DOI: 10.1021/acs.jafc.7b02748 J. Agric. Food Chem. 2017, 65, 8331−8339
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Journal of Agricultural and Food Chemistry
shown in Table 4, the highest degradation rate constant of JuA was found in the gastric contents (0.34 h−1), which is almost equal to that in artificial gastric juice (0.35 h−1). This indicating pH might play a key role in the degradation. The degradation rate constant of JuA in the colon contents was 0.06 h−1. This was much lower than that in the stomach and duodenum, but was slightly higher than that in the jejunum and ileum, indicating that the saponin was relatively stable in such an environment, i.e. even only a smaller degree of degradation occurred. Identification of the Metabolites. In this study, over 10 metabolites were found after JuA was incubated with gastric contents. As shown in Figure 5, three main metabolites (M1, M2, and M3) with high quantity were identified. The m/z of the excimer ion [M − H]− of M1 was 1043.5, suggesting that it might lose one glucose (162.1) from the parental structure (1205.6). The fragment ions generated in the secondary mass spectrum were 911.2 and 749.5. M1 was further identified as jujuboside B based on its chromatographic behavior and mass spectrometry characters. M3 was characterized as jujubogenin, resulting from the loss of two molecules of glucose, one xylose, one arabinose, and one rhamnose. Effects of the Metabolites on Hippocampal Neurons. The hippocampal neurons (being cultured for 8 days) exhibited a characteristic shape: the obvious cell body, single long axon, and several shorter tapering dendrites. MAP2 immunofluorescence staining showed that the neurons and protrusions were green fluorescence, while the nucleus was stained blue. The purity of the neurons was measured to be over 90% (Figure 6A). When the hippocampal neurons were treated with different concentrations of JuB and jujubogenin for 24 h, the cell viability was increased compared with the blank control group, and the high concentration of JuB showed a significant difference (Figure 6B). The results indicated that JuB played a significant role in promoting the growth of hippocampal neurons. However, jujubogenin did not exhibit the same effect. The increase of the GABA(A) α1 and GABA(A) α5 relative expression level could be induced by the two metabolites in a dose dependent manner (Figure 6C). In addition, the average MQAE fluorescence intensity of chloride ion in hippocampal cells treated with JuB and jujubogenin was significantly lower than that of the control group (Figure 6D). The results indicated that the two metabolites could increase the influx of chloride ion obviously, and the effect was similar to that of diazepam.
Figure 3. Intestinal absorption of JuA in the duodenum, jejunum, ileum, and colon. (A) The absorbed trend of JuA with the initial concentration 20 μg·mL−1 within 4 h in each bowel. (B) Histogram of the absorption percentage of each intestinal segment. Values are in mean ± SD (n = 3). *p < 0.05 and **p < 0.01 compared to the maximum value (duodenum).
Metabolic Dynamics of JuA. The calibration equation and the regression coefficients (R2) of the calibration curves are summarized in Table 3, which all showed good linearity within Table 3. Regression Equations and Correlation Coefficients of the Standard Curve of JuA in Each Kind of Incubation Solution Incubation solution types Sucrose solution (0.3 M) Kreb-Ringer’s buffer solution HAc/NaAc buffer solution (1 M) HCl solution (pH 1.2) Artificial gastric juice Artificial intestinal fluid
Regression equation Y Y Y Y Y Y
= = = = = =
327.48X 367.44X 394.24X 390.02X 514.49X 504.22X
+ 232.20 − 58.92 + 152.14 − 31.76 − 55.818 − 158.31
R2 0.9941 0.9985 0.9978 0.9999 0.9965 0.9994
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DISCUSSION JuA is believed to be the main constituent responsible for the anxiolytic and sedative effect of ZSS. Studies have shown that JuA can exert its specific bioactivity through various mechanisms.33 However, some evidence implicates that JuA may not be the real active form, in spite of the fact that this hypothesis has not been demonstrated experimentally. Here, for the first time, we revealed that the bioavailability of JuA was very low, which was not caused by its malabsorption in the intestine but the metabolic effect of the gastrointestinal tract. Our findings provide new insight into the mechanisms of JuA bioactivity. In this study, the bioavailability of JuA in rats was determined as 1.32%, suggesting that only a trace amount of JuA was absorbed into the body. Apparently this result was similar to the fate of other saponins, such as ginsengsaponin,34
the concentration range. The results of the intra- and interday precision, accuracy, extraction recovery, and matrix effect of JuA in all the QC samples are summarized in Table S2 (Supporting Information). The results suggested that the present method was reproducible and accurate for the determination of the analyts in the culture solution. The degradation course of JuA in gastric environment, different intestinal segments, artificial gastric juice, and aitificial intestinal fluid is shown in Figure 4. It was found that the concentration of JuA decreased with the prolongation of time from the degradation curves. Overtly, the degradations of JuA in the gastric environment and artificial gastric juice were pronounced, suggesting that JuA is vulnerable to such an acidic environment. To determine the effect of the environment on JuA degradation, the degradation rate constant of JuA was calculated in the presence of various microenvironments. As 8335
DOI: 10.1021/acs.jafc.7b02748 J. Agric. Food Chem. 2017, 65, 8331−8339
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Figure 4. Degradation curves of JuA in different incubation solutions. (A) Gastric environment. (B) Duodenal environment. (C) Jejunal environment. (D) Ileal environment. (E) Colonic environment. (F) Artificial gastric juice and artificial intestinal fluid.
Table 4. Degradation Rate Constants of JuA in Each Incubation Solution (n = 3) The degradation rate constants (h−1) Incubation solution types Contents Cytosol BBM Lysosome
Stomach 0.34 0.22 0.23 0.21
± ± ± ±
0.02 0.03 0.01 0.03
Duodenum 0.17 0.15 0.08 0.09
± ± ± ±
0.04 0.04 0.02 0.01
Jejunum 0.03 0.02 0.02 0.01
± ± ± ±
0.01 0.00 0.00 0.00
Ileum 0.03 0.05 0.01 0.01
± ± ± ±
0.00 0.01 0.00 0.00
Colon
Artificial gastric juice
Artificial intestinal fluid
± ± ± ±
0.35 ± 0.02
0.01 ± 0.00
0.06 0.01 0.04 0.01
0.01 0.00 0.01 0.00
Figure 5. Metabolic pathway of JuA in rat gastric contents.
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Figure 6. Effects of the metabolites on the expression and activation of GABA(A) in rat hippocampal neurons. (A) The hippocampal neurons being cultured for 8 days: (a) Representative inverted micrograph (10 × 4); (b) Microscopic images of nucleus DAPI staining (10 × 4); (c) Microscopic images of MAP-2 positive cells (10 × 4). (B) Cell viability of hippocampal neurons treated with JuB and jujubogenin. (C) The relative expression levels of GABA(A) α1 and GABA(A) α5 receptors under the treatment of JuB, jujubogenin, and diazepam (positive control group). (D) The variational histogram of the MQAE fluorescence intensity of chloride ion. Values are mean ± SD (n = 6). *p < 0.05 compared to blank control group (A, C) or positive control group (B).
kalopanaxsaponin A,35 astragaloside III,36 and escin Ia.37 The reasons for the poor oral bioavailability of JuA may result from poor absorption, acidic/basic instability, extensive microbial and hepatic metabolism, etc. Then the absorption of JuA in the different intestinal segments of the rats was examined to confirm its bioaccessibility. Surprisingly JuA can be well absorbed in rat intestine, implicating its poor bioavailability is not caused by malabsorption.38 In addition, the absorption rate constant and percentage in the duodenum were significantly higher than that in the other parts of the intestine, suggesting that the duodenum is the main site for absorption of JuA. Interestingly, the absorptive capacity of the colon was higher than those of the ileum and jejunum. This might be due to the specific metabolism associated with the micro-organisms in the colon. To further verify the results, the level of JuA in the blood samples collected from the rat hepatic portal vein was determined at the same time. It can be inferred that the duodenum and colon are the better absorption sites. This conclusion was consistent with the in situ perfusion of intestinal experiments. Furthermore, the plasma concentration of JuA in rats was investigated at the end of the experiment, and the results showed that it can be found at a fair level. This indicated that the first pass effect of the liver could not metabolize JuA totally (data not displayed). The elimination rate of JuA in the stomach, including its contents and mucosal subcellular organelles, was high, indicating that the gastric environment had a significant effect on its degradation. Obviously gastric acid might play a critical role in hydrolysis of JuA because artificial gastric juice (pH 1.2) exhibited a similar effect on JuA degradation. In addition, the
gastric mucosal subcellular organelles also contributed to this process. To some extent, all contents of intestinal segments, including the duodenum, jejunum, ileum, colon, and their mucosal subcellular organelles, were found to be involved in the degradation of JuA. Among them, the duodenum contents displayed superior degradation capacity, which may be attributed to its specific condition in the duodenum. JuA is a triterpenoid glycoside linked with 5 sugar groups (2 glucoses, 1 rhamnose, 1 xylose, and 1 arabinose), which are bound to the aglycone through various valence bonds, such as β-D-xyl-β-D-glc, β-D-glc-β-D-glc, and α-L-ara-β-D-glc. After incubation with gastric contents, over 10 metabolites were detected. Through analyzing the molecular ions and product ions in MS spectra, JuB and jujubogenin were identified as the main products of the saponin hydrolysis. In our previous report, the saponin could also be degraded to form 7 metabolites by rat intestinal flora,23 in which JuB and jujubogenin were also the main hydrolysis products, suggesting that it may be the real absorbed form with the specific bioactivity. The sedative-hypnotic effects of JuB and jujubogenin were investigated in hippocampal neurons with high purity and typical characteristics. Cell viability assay showed that JuB had a certain proliferative effect on the neurons. GABA(A) α1 was mainly involved in the sedative-hypnotic effect,39 while the GABA(A) α5 subunit mediated muscle relaxation mainly, which could accelerate the occurrence of the sedative-hypnotic effect. The promoting effect of JuB and jujubogenin on the GABA(A) α1 and GABA(A) α5 expression levels suggested that the sedation-hypnotic effect might be achieved primarily by increasing the number of GABA(A) receptors on the 8337
DOI: 10.1021/acs.jafc.7b02748 J. Agric. Food Chem. 2017, 65, 8331−8339
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Journal of Agricultural and Food Chemistry
(3) Ma, Y.; Han, H.; Nam, S. Y.; Kim, Y. B.; Hong, J. T.; Yun, Y. P.; Oh, K. W. Cyclopeptide alkaloid fraction from Zizyphi Spinosi Semen enhances pentobarbital-induced sleeping behaviors. J. Ethnopharmacol. 2008, 117, 318−324. (4) Yi, P. L.; Lin, C. P.; Tsai, C. H.; Lin, J. G.; Chang, F. C. The involvement of serotonin receptors in suanzaorentang-induced sleep alteration. J. Biomed. Sci. 2007, 14, 829−840. (5) Xie, J.; Qiao, L. D.; Song, M. Y.; Wang, L. J.; Zhang, Y. Q.; Feng, H. HPLC-ESI-MS/MS analysis of the water-soluble extract from Ziziphi Spinosae Semen and its ameliorating effect of learning and memory performance in mice. Pharmacogn. Mag. 2014, 10, 509−516. (6) Xie, J. B.; Zhang, Y. Q.; Wang, L. J.; Qi, W. Q.; Zhang, M. C. Composition of fatty oils from Semen Ziziphi Spinosae and its cardiotonic effect on isolated toad hearts. Nat. Prod. Res. 2012, 26, 479−483. (7) Ma, Y.; Han, H.; Eun, J. S.; Kim, H. C. Sanjoinine A isolated from Zizyphi Spinosi Semen augments pentobarbital-induced sleeping behaviors through the modification of GABA-ergic systems. Biol. Pharm. Bull. 2007, 30, 1748−1753. (8) Yingjun, L.; Shou, C. Application of Overexcitation model induced by penicillin sodium in the study of inhibitory effect of sedative hypnotic drugs. Pharm. Biol. 2005, 43, 308−312. (9) Cao, J. X.; Zhang, Q. Y.; Cui, S. Y.; Cui, X. Y.; Zhang, J. A.; Zhang, Y. H.; Bai, Y. J.; Zhao, Y. Y. Hypnotic effect of jujubosides from semen Ziziphi Spinosae. J. Ethnopharmacol. 2010, 130, 163−166. (10) Shou, C.; Feng, Z.; Wang, J.; Zheng, X. The inhibitory effects of jujuboside A on rat hippocampus in vivo and in vitro. Planta Med. 2002, 68, 799−803. (11) Zhang, M.; Ning, G. G.; Shou, C. H.; Lu, Y. J.; Hong, D. H.; Zheng, X. X. Inhibitory effect of jujuboside A on glutamate-mediated excitatory signal pathway in hippocampus. Planta Med. 2003, 69, 692− 695. (12) You, Z. L.; Xia, Q.; Liang, F. R.; Tang, Y. J.; Xu, C. L.; Huang, J.; Ling, Z. B.; Zhang, W. Z.; He, J. J. Effects on the expression of GABAA receptor subunits by jujuboside A treatment in rat hippocampal neurons. J. Ethnopharmacol. 2010, 128, 419−423. (13) Wang, X. X.; Ma, G. J.; Xie, J. B.; Pang, G. C. Influence of JuA in evoking communication changes between the small intestines and brain tissues of rats and the GABA A, and GABA B, receptor transcription levels of hippocampal neurons. J. Ethnopharmacol. 2015, 159, 215−223. (14) Chen, C. Y.; Chen, Y. F.; Tsai, H. Y. What is the effective component in suanzaoren decoction for curing insomnia? Discovery by virtual screening and molecular dynamic simulation. J. Biomol. Struct. Dyn. 2008, 26, 57−64. (15) Chen, C. Y. Insights into the suanzaoren mechanismFrom constructing the 3D structure of GABA-A receptor to its binding interaction analysis. J. Chin. Inst. Chem. Eng. 2008, 39, 663−671. (16) Liu, C.; Li, Y.; Zhong, Y.; Huang, X.; Zheng, X.; Li, N.; He, S.; Mi, S.; Wang, N. An LC-MS/MS method for determination of jujuboside A in rat plasma and its application to pharmacokinetic studies. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2012, 899, 21−26. (17) Felgines, C.; Fraisse, D.; Besson, C.; Vasson, M. P.; Texier, O. Bioavailability of lemon verbena (Aloysia triphylla) polyphenols in rats: impact of colonic inflammation. Br. J. Nutr. 2014, 111, 1773− 1781. (18) Li, S. X.; Chen, L. H.; Zheng, F. Y.; Li, Y. C. Effect of the cp4epsps gene on metal bioavailability in maize and soybean using bionic gastrointestinal tracts and ICP-MS determination. J. Agric. Food Chem. 2013, 61, 1579−1584. (19) Domínguez-Avila, J. A.; Wall-Medrano, A.; VelderrainRodríguez, G. R.; Chen, C. O.; Salazar-López, N. J.; Robles-Sánchez, M.; González-Aguilar, G. A. Gastrointestinal interactions, absorption, splanchnic metabolism and pharmacokinetics of orally ingested phenolic compounds. Food Funct. 2017, 8, 15−38. (20) Dickie, A. P.; Wilson, C. E.; Schreiter, K.; Wehr, R.; Wilson, E. M. The pharmacokinetics and metabolism of lumiracoxib in chimeric
membrane surface of the hippocampal neurons, which provides a basis for the combination therapy in the treatment of insomnia and anxiety disorders. Furthermore, we used the MQAE fluorescence probe technique to evaluate the chloride ion channel activation of GABA(A) receptors. The results showed that the sedative and hypnotic mechanism of JuB and jujubogenin might be similar to that of diazepam, which could increase the opening frequency of the chloride ion channel, thus playing to the anxiolytic sedation effect.40 Taken together, the absorption and metabolic dynamics of JuA were investigated in vivo and in vitro for the first time in the present study. Our findings demonstrate that the metabolites of the saponin, not the original molecule, should be responsible for the sedative bioactivities.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b02748. Nucleotide sequence of the forward and reverse primers; Precision, accuracy, extraction recovery and matrix effect of JuA in sample solutions (n = 3) (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*Tel.: +862226667633. Fax: +862226686254. E-mail address:
[email protected] (Y. Q. Zhang). *E-mail address:
[email protected] (J. B. Xie). ORCID
Panpan Song: 0000-0002-6030-284X Junbo Xie: 0000-0002-1666-3584 Funding
This work was supported by the National Natural Science Foundation of China (Grant No. 31101235; No.31000749) and “131” innovative talents training project in Tianjin. Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED JuA, Jujuboside A; JuB, Jujuboside B; ZSS, Zizyphi Spinosae Semen; GABA(A), γ-amino-butyric acid A; AUC, the area under the plasma concentration time curve; RTFQ-PCR, real time fluorescence quantitative reverse transcription polymerase chain reaction; MQAE, N-ethoxycarbonylmethyl-6-methoxyquinolinium bromide; MS, mass spectrometry; SD, Sprague− Dawley; ESI, electrospray ionization; MRM, multiple reaction monitoring; [M − H]−, the characteristic transitions of precursor; Cmax, the maximum plasma concentration; Tmax, the time to reach Cmax; t1/2, the elimination half-life; Ke, the elimination rate constant; F, bioavailability; BBM, the brush border membrane; Ka, the absorption rate constant; PBS, phosphate buffer solution
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