Mangiferin (MG) is an active component in natural medicines, and various studies have been reported on pharmacological effects, but the low solubility and bioavailability of MG limit its wide application. The aim of the present study was to investigate the pharmacokinetic profiles of mangiferin (MG) and mangiferin monosodium salt (MG-Na) in rat plasma by UPLC-MS/MS, which were then compared between the two groups. An appropriate high sensitivity and selectivity ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was applied to the comparison of plasma pharmacokinetics in MG and MG-Na using carbamazepine as internal standard (IS). These results showed that there were statistically significant differences in the pharmacokinetic parameters between MG and MG-Na after a single oral administration at 100 mg/kg. When compared with pharmacokinetic parameters of MG, the AUC(0-
Mangiferin (MG, Figure
Chemical structure of MG and MG-Na.
Recently, researchers have carried out a series of structural modifications on MG, such as alkylation reaction, acylation reaction, a salification reaction, and phospholipidation reaction [
For further research and development of MG-Na, we systematically studied the pharmacokinetic comparison of MG and MG-Na rats in vivo in the present study [
MG sample (C16H12O4, purity >98.00%) and MG standard sample (purity >99.99%) were purchased from Chroma Biological Co. Ltd. (Sichuan, China). Carbamazepine (C15H12N2O, IS) was obtained from the National Institutes for Food and Drug Control (Beijing, China). Methanol and acetonitrile (HPLC grade) were purchased from Fisher Scientific (Pittsburgh, PA, USA). Formic acid of HPLC grade was supplied from Aladdin Industrial Co. Ltd. (Shanghai, China). Acetone (CH3COCH3) and sodium bicarbonate (NaHCO3) were purchased from Kermel Chemical Reagent Co. Ltd. (Tianjin, China). Heparin sodium was obtained from Wanbang biochemical Medicine Group Co. Ltd. (Jiangsu, China). Ultrapure water (sensitivity of 18.80 MΩ) was prepared by passing distilled water with a Milli-Q system (Millipore, Shanghai, China).
The synthesis and purification of MG-Na were performed in our laboratory involved method (Figure
Forty male Sprague Dawley (SD) rats (7 weeks old, weighing 200 ± 20 g) and eighty Kunming mice with both sexes (7 weeks old, weighing 20 ± 2 g) were provided by the Vital River Laboratory Animal Technology (Beijing, China; certification number: 11400700243083 and 1100111911036181; license no. SCXK (Jing) 20160006 and 20160011). Ethical approval for the experimental protocols was obtained from the Animal Ethical and Welfare Committee (AEWC) of Hebei University (approval number: IACUC-2018045 dated 11/05/2018). All experimental rats were fed one week before the experiment. The rats were housed in polycarbonate cage and kept at constant temperature (23 ± 2°C) and relative humidity (65 ± 5%) with a 12 h light/dark cycle. The rats had free access to standard chow diet and water
In the study, the rats were selected to perform the pharmacokinetic comparisons of MG and MG-Na according to previous reports and ICH guidelines by European Medicines Agency [
The stock solutions of MG and MG-Na were prepared by dissolving accurately weighed quantity of each drug in methanol at the concentration of 0.1 mg/mL. The above standard stock solutions were diluted with methanol to obtain standard working solutions at concentrations of 10.0, 20.0, 50.0, 100.0, 200.0, 500.0, 1000.0, 2000.0, and 6000.0 ng/mL for MG and MG-Na, respectively. Stock solution for IS was prepared at the concentration of 0.1 mg/mL in methanol and diluted with methanol to yield the working solution of IS at 100 ng/mL. The calibration standards were prepared by adding a series of 10
Plasma samples were prepared by adding 10
The ultraperformance liquid chromatography (UPLC) system was connected with a Vanquish System (Thermo Scientific, San Jose, CA, USA) which consists of two pumps, a detector, and a sample room. The chromatographic column was ACQUITY UPLC BEH-C18 column (1.7
Mass spectrometric detection was performed on a TSQ-Altis-10265 mass spectrometer (Thermo Scientific, San Jose, CA, USA) equipped with an electrospray ionization interface (ESI). The plasma samples were detected by a TSQ Altis MS/MS system in the ESI negative ion mode. Following the optimization of mass spectrometric parameters, the positive ion voltage, negative ion voltage, sheath gas, auxiliary gas, sweep gas, ion transfer tube temperature, and vaporizer temperature were set at 3500 V, 3500 V, 50 Arb, 23.2 Arb, 0 Arb, 52°C, and 350°C, respectively. The selective reaction monitoring (SRM) was applied for the detection of MG, MG-Na, and IS transition of
The specificity was performed by analyzing six different drug-free blank plasma samples, drug-free blank plasma spiked with analytes and IS, and drug-free blank plasma of the rats after oral administration of analytes (MG and MG-Na), respectively. Chromatographic review was carried out to assess the potential endogenous interference, which developed a method can be considered to have acceptable selectivity if no interfering endogenous substances were observed at the retention times of the analytes and IS.
Linear calibration curves in blank plasma were drawn by plotting the peak area ratios (Y) of the analytes (MG and MG-Na) to the IS vs. the concentrations (
The precision and accuracy were analyzed by the QC samples at three concentrations (3, 160, and 480 ng/mL). The intraday and interday precision and accuracy were determined by analyzing six replicates of plasma samples on the same day and three consecutive days, respectively. The standard curve was used to calculate the concentration of each plasma sample on the same day. Furthermore, the precision and accuracy were evaluated by the relative standard deviation (RSD, <15%) and RE (±15%), respectively. The acceptance criterion of RSD should be less than 15%, and the acceptance standard of RE should be within ±15%.
To investigate the extraction recovery and matrix effects, QC samples from 6 replicates at three concentration levels (3, 160, and 480 ng/mL) were analyzed. The extraction recovery of two analytes was analyzed by comparing the peak areas of analytes (MG and MG-Na) spiked before precipitated protein with those of analytes spiked after precipitated protein. And, the matrix effect was measured by comparing the peak of analytes spiked after precipitated protein with those of analytes in reconstitution solution. These procedures were repeated for six replicates at three QC concentration levels, and the results were required to be within ±15%.
The stability of MG and MG-Na in rat plasma during sample storing and processing procedures was evaluated by six replicates of QC samples under different conditions, such as short-term stability, long-term stability, and freeze-thaw stability. The short-term stability was analyzed after the storage of QC samples exposed to room temperature for 6 h, 12 h, and 24 h. The long-term stability was investigated after the storage of QC samples at −80°C for 4 weeks. To evaluate the freeze-thaw stability, the QC samples were measured after three cycles of freeze (−80°C) and thaw (20°C).
To evaluate the dilution procedure, the dilution integrity of analytes was performed by four different concentrations of MG and MG-Na diluting a high concentration of plasma samples (1000 ng/mL) with blank plasma to a low concentration (10, 100, and 500 ng/mL). The diluted samples were analyzed with six batches by a freshly prepared calibration curve. The RE of each plasma sample should be within ±15%, and the RSD of each plasma sample should not exceed 15%.
Carryover of the tested analytes was assessed by injection of drug-free blank plasma samples after injection of the upper limit of quantification (ULOQ, 600 ng/mL). The measured peak area should be within 20% of the MG and MG-Na peak area at the LLOQ level.
The rats (
According to the above calibration curves, the concentration of drug was calculated in plasma of all rats at each time point. The pharmacokinetic parameters of the analytes (MG and MG-Na) were automatically simulated and calculated by utilizing Drug and Statistics (DAS) 2.0 (Chinese Pharmacological Society). The elimination constant (
The results were presented as mean ± SD. Statistical analysis between MG and MG-Na groups was statistically analyzed and processed using SPSS 16.0 (Statistical Package for the Social Science) by independent samples
Recently, there were several reports on the LC, HPLC, and HPLC-MS analysis of MG in plasma [
To investigate the pharmacokinetics of MG-Na, we first performed the synthesis of MG-Na in the present study. MG-Na was synthesized by the reaction of mangiferin with sodium bicarbonate. The product was yellow powder with a yield of 81.81%. The purity of MG-Na was verified by UPLC to be more than 93.50% and MG-Na was purified to 98.00%. Here, the elementary analysis results of MG-Na showed that Anal. Calcd. for C19H17O11Na: C, 51.35; H, 3.83; O, 39.64; Na, 5.18%. Found: C, 51.20; H, 3.23; O, 39.87; Na, 5.09%. In addition, our results showed that the water solubility of MG-Na was greatly improved compared with that of the MG in this study.
As shown in Figure
The typical equation of calibration curves and linearity ranges for the analytes (MG and MG-Na) are shown in Figure
The results of the precision and accuracy of the methods are shown in Table
Precision and accuracy of MG and MG-Na in rat plasma (
Plasma concentration | Intraday | Interday | ||||
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Mean concentration | RSD (%) | RE (%) | Mean concentration | RSD (%) | RE (%) | |
MG | ||||||
3 | 3.172 ± 0.152 | 4.7 | 4.5 | 2.933 ± 0.118 | 4.0 | −2.2 |
160 | 166.357 ± 10.674 | 6.4 | 3.9 | 168.537 ± 11.731 | 6.9 | 5.3 |
480 | 486.523 ± 13.543 | 2.9 | 1.4 | 483.545 ± 23.848 | 4.9 | 07 |
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MG-Na | ||||||
3 | 3.124 ± 0.107 | 3.4 | 4.1 | 3.201 ± 0.179 | 5.6 | 6.7 |
160 | 168.343 ± 8.472 | 5.0 | 5.2 | 172.498 ± 10.549 | 6.1 | 7.8 |
480 | 491.573 ± 10.456 | 2.1 | 2.4 | 489.384 ± 16.331 | 3.3 | 2.0 |
The results of the absolute extraction recoveries and matrix effects in the above QC samples (3, 160, and 480 ng/mL) are presented in Table
Extraction recovery and matrix effects of MG and MG-Na in rat plasma (
Plasma concentration | Extraction recovery rate | Matrix effect | ||||
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Recovery rate (%) | RSD (%) | RE (%) | Matrix effect (%) | RSD (%) | RE (%) | |
MG | ||||||
3 | 96.6 ± 4.2 | 4.2 | −3.4 | 97.1 ± 3.4 | 3.4 | −2.9 |
160 | 103.5 ± 2.7 | 2.7 | 3.5 | 98.2 ± 2.3 | 2.3 | −1.8 |
480 | 97.5 ± 3.3 | 3.3 | −2.5 | 98.5 ± 1.7 | 1.7 | −1.5 |
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MG-Na | ||||||
3 | 97.1 ± 2.8 | 2.8 | −2.9 | 98.3 ± 3.1 | 3.1 | −1.7 |
160 | 98.6 ± 1.1 | 1.1 | −1.4 | 99.1 ± 1.3 | 1.3 | −0.9 |
480 | 102.7 ± 2.3 | 2.3 | −2.7 | 98.2 ± 2.4 | 2.4 | −1.8 |
The stability results of MG and MG-Na at QC samples (3, 160, and 480 ng/mL) in plasma sample are presented in Tables
Stability of MG in rat plasma (
Plasma concentration | Measured concentration | RSD (%) | RE (%) |
---|---|---|---|
Short-term stability | |||
3 | 3.166 ± 0.192 | 6.1 | 5.5 |
160 | 153.591 ± 9.592 | 6.2 | −4.0 |
480 | 4486.328 ± 25.495 | 5.2 | 1.3 |
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Long-term stability | |||
3 | 3.147 ± 0.201 | 6.4 | 4.9 |
160 | 169.553 ± 11.295 | 6.7 | 6.0 |
480 | 494.472 ± 31.491 | 6.4 | 3.0 |
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Freeze-thaw stability | |||
3 | 3.127 ± 0.112 | 3.6 | 4.2 |
160 | 154.212 ± 6.521 | 4.2 | −3.6 |
480 | 473.351 ± 17.381 | 3.7 | −1.4 |
Stability of MG-Na in rat plasma (
Plasma concentration | Measured concentration | RSD (%) | RE (%) |
---|---|---|---|
Short-term stability | |||
3 | 3.053 ± 0.121 | 4.0 | 1.8 |
160 | 171.937 ± 9.376 | 5.5 | 7.5 |
480 | 496.593 ± 15.118 | 3.0 | 3.5 |
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Long-term stability | |||
3 | 3.198 ± 0.193 | 6.0 | 6.6 |
160 | 163.518 ± 8.574 | 5.2 | 2.2 |
480 | 487.412 ± 21.382 | 4.4 | 1.5 |
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Freeze-thaw stability | |||
3 | 3.209 ± 0.214 | 6.7 | 7.0 |
160 | 152.298 ± 8.418 | 5.5 | −4.8 |
480 | 469.552 ± 19.317 | 4.1 | −2.2 |
Dilution integrity is necessary when the concentration of analytes in the samples is expected to over the ULOQ. The dilution integrity was determined by analyzing 6 replicates of MG and MG-Na in Table
In the current study, carryover was assessed via injection of processed drug-free blank samples followed by the ULOQ. The results of the carryover are shown in Table
The above-validated UPLC-MS/MS method was successfully applied to the plasma comparative pharmacokinetic profiles of MG and MG-Na after a single oral administration at 100 mg/kg in rats in vivo, respectively. The mean concentration-time curves of MG and MG-Na in plasma were analyzed by utilizing Drug and Statistics (DAS) 2.0 software to determine the compartment model, and the results are shown in Figures
The plasma concentration-time curve of MG and MG-Na. The C-T curve after oral administration of MG (a) and the C-T curve after oral administration of MG-Na (b).
Mean compartmental pharmacokinetic parameters of MG and MG-Na (
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CL/ |
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MG | 60.00 ± 0.00 | 23.878 ± 4.457 | 69.315 ± 11.782 | 7565.692 ± 1731.293 | 7600.169 ± 1687.238 | 0.002 ± 0.000 | 0.022 ± 0.001 | 13.158 ± 1.589 | 2693.381 ± 355.336 | 100 |
MG-Na | 15.00 ± 0.00 |
496.867 ± 79.472 |
72.315 ± 10.652 | 42493.728 ± 580.387 |
42983.947 ± 674.291 |
0.009 ± 0.001 |
1.84 ± 0.129 |
2.326 ± 0.448 |
248.442 ± 20.351 |
570 |
As summarized in Figures
The result of
As shown in Table
Overall, big changes are being observed in the mean plasma concentration-time profiles and pharmacokinetic parameters between MG and MG-Na after a single oral administration, which suggested that the structural modification by a salification reaction of MG induced a remarkable enhancement in gastrointestinal absorption and relative bioavailability of MG by improving solubility and membrane permeability in the present study. Thus, the above pharmacokinetics study of MG-Na may be more helpful for the farther development and clinical study of MG-Na in the near future. Additionally, more research studies are needed to clarify the functional mechanisms of the oral relative bioavailability increase of MG-Na.
Single oral doses of MG or MG-Na 400 mg were generally safe for healthy rat subjects when administered orally in the fed or fasted state (data not shown). No deaths, serious adverse events, or other significant adverse events were found during the study. At the same time, the MG and MG-Na have no safety concerns by the parameter assessment of behavior (walk and sleep), food, water and energy intake, hair, body weight, tissue weight, liver function (ALT, AST, and ALT/AST), liver histopathology, and feces and urine color.
In this study, the toxicity studies of MG-Na (such as LD50 and maximum tolerated dose (MTD)) were performed by gavage (data not shown). Here, mice rather than rats were used because it is scientifically documented that lethal dose data collected from mice might be more appropriate to anticipate the toxic effects in human beings [
The above results suggested that MG-Na after oral administration is safe and well tolerated and has no toxicity, which provides a certain security guarantee for MG-Na study and development in the near future.
The numerous clinical experiments show that the absorption, distribution, metabolism, excretion, and toxicity process of drugs are important indicators of druggability. According to the physical properties of drug candidates, the drug structure will be designed rationally and this study illustrates this fact by transforming poorly soluble MG into MG-Na with good solubility. The result of main pharmacokinetic comparisons of MG and MG-Na showed that the pharmacokinetic parameters of MG and MG-Na have remarkable differences, which suggested that the salification reaction of MG can effectively enhance gastrointestinal absorption and relative bioavailability by improving solubility and membrane permeability. To our knowledge, this is the first report demonstrating pharmacokinetic comparisons of mangiferin and mangiferin monosodium salt in rat plasma by UPLC-MS/MS. Simultaneously, the resulting pharmacokinetic data can aid the understanding of the safety of MG-Na and lay the foundation for future drug research.
The data used to support the findings of this study are included within the article. Data are available from the corresponding author (Chengyan Zhou,
The authors declare no conflicts of interest.
C.Y.Z. designed the experiments and reviewed the manuscript before submission. G.H.B. performed the data analysis and discussed the results. C.M.Q. performed analyses and drafted the manuscript. L.M.R. performed the research. C.B.H. substantially contributed to the literature search and statistical analyses. All authors read and approved the final manuscript for publication.
This research was supported by the project from the Science and Technology Research and Development Guidance Plan of Baoding City (no. 18ZF121) and College Students Innovation and Entrepreneurship Training Program of Hebei University (no. 2018280).
The supplementary materials consist of four figures and three tables to further clarify the method validation, synthesis procedures for MG-Na, and pharmacokinetic comparison results of MG and MG-Na in rat plasma.