The purpose of this study is to investigate the effects of Borneol on the pharmacokinetics of notoginsenoside R1 (NGR1) and the ginsenosides Rg1 (GRg1) and Re (GRe) in
Various methods for the quality control of
Borneol, a monoterpenoid component of the medicinal plant such as
The current study is to investigate the effect of Borneol on the pharmacokinetics of NGR1, GRg1, and GRe in
NGR1, GRg1, and GRe (purity > 95%) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products of China (Lots nos. 110754-200322; 110703-200322; and 110745-200414, resp.). Borneol (purity > 98%) was supplied by Tianjin Tasly Pharm. Co., Ltd. Caco-2 cells were acquired from Institute of Biochemistry and Cell Biology, Shanghai institute for Biological Sciences, CAS. Transwell plates (pore size 0.4
250 grams of
The ethical use of animals in this study was approved by the Advisory Board on Animal Experiments of the Xi’an Jiaotong University in China. New Zealand rabbits (weight 1.7–2.3 kg) were provided by the Animal Center of Xi’an Jiaotong University. The rabbits were maintained in air-conditioned animal quarters at a temperature of 22 ± 2°C and a relative humidity of 50 ± 10%. The cannula (Terumo, 22 G × 1, i.d. 0.60 × 20 mm) was placed in the central ear artery and used for blood collection. The animals were acclimatized to the facilities for 5 days, and then fasted and had free access to water for 12 h prior to experiment.
Liquid chromatography was carried out on an Agilent 1100 HPLC system with an auto sampler, a quaternary pump and a vacuum degasser (Waldoboro, Frankfurt, Germany). Operations were controlled by Agilent Chemstation 4.2 software (Littleforts, Philadelphia, USA). Separations were achieved on a reversed-phase HPLC column (Zorbax SB-C18 150
MSn detection was performed on an Agilent SL trap MS system (Waldoboro, Frankfurt, Germany). The ion source-dependent (electrospray ionization) conditions were the same for all analyses with a spraying voltage of −4500 V in the negative ion mode. The pressure of the nebulizing gas (nitrogen) was set at 35 p.s.i. The flow rate of the drying gas (nitrogen) was set at 7.0 L·min−1 with the temperature of 325°C. The collision gas (He) for the MSn mode at trap was set at flow of 4 (instrument unit). The voltage of the capillary was set at 4000 V, and its end plate offset was −500 V. Scan range was from 500 to 1500
Primary stock solutions of 0.28 mg·min−1 NGR1, 0.30 mg·min−1 GRg1 and 0.72 mg·min−1 GRe were prepared in methanol. Working standard solutions of NGR1, GRg1, and GRe were prepared by diluting the aliquots of the primary solution with methanol. The solutions were stored at 4°C in glass tubes until further use.
Frozen plasma and tissue samples were thawed in a water bath at 37°C and were then vortexed followed by centrifuging at 5000 r·min−1 for 5 min. An aliquot of 1.0 mL of the supernatant from each sample was loaded onto C18 Bond Elute Solid phase extraction (SPE) cartridges (1000 mg, 1 cc reservoir, Varian, Harbor City, CA, USA) pretreated with 2.0 mL hexane, isopropanol, methanol, and water, sequentially. The SPE cartridges were then washed with 1.0 mL water, 20% methanol/water solution, 40% methanol/water solution, and 60% methanol/water solution, sequentially. Finally, analytes were eluted twice with 1.0 mL of 70% methanol/water solution. The eluant was evaporated to dryness under nitrogen. The residues were then reconstituted in 1.0 mL mobile phase. An aliquot of 10
Samples calibration standards containing 0.28, 0.56, 2.8, 5.6, 14.0, 28.0, and 56.0
Validation of the proposed method included assessment of the calibration curve performance, as well as accuracy and precision of the method, and stability of the analytes at various test conditions.
The precision of the assay was determined for the quality control (QC) plasma and tissue samples by replicate analyses of three levels of concentration at 0.5, 5.0, and 35.0
Eighteen rabbits were randomly divided into three groups of 6 subjects and were orally given 3.0 mL·kg−1 normal saline, 3.0 mL·kg−1
One group of rabbits (
The Caco-2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% foetal bovine serum, 1% nonessential amino acids and penicillin-streptomycin, at 37°C in an atmosphere with a relative humidity of 95% and a CO2 flow of 5%. Medium was replaced every 2-3 days. When the cell monolayer reached 80% confluence, the cells were detached with a solution of 0.02% EDTA and 0.25% trypsin. The vinblastine-selected Caco-2 cells were cultivated in the presence of 10 nM vinblastine to induce P-glycoprotein (P-gp) expression. The culture medium was changed to a fresh medium without vinblastine 24 h before experiments, and the cells were used between passages 25 and 46. Prior to the transport study, cytotoxicity of NGR1, GRg1, GRe, and Borneol toward Caco-2 cells was determined using MTT assays. Noncytotoxic concentrations of 500
In transport studies, vinblastine-selected Caco-2 cells were seeded on polycarbonate filter of transwells for 18–21 days before starting transport study, and the monolayers with the transepithelial electrical resistance (TEER) values greater than 300
The efflux ratio (
Statistical analysis of the biological data was performed using the Student’s
The base peaks of each mass spectrum for NGR1, GRg1, and GRe were observed during the infusion of the standard solution in negative mode. Three
HPLC-MS ion chromatograms of plasma samples. (a) blank plasma samples; (b) plasma standard solutions of 5.0
The calibration curves were created by plotting the peak areas of NGR1, GRg1, and GRe to their various concentrations in the spiked plasma and tissue standards. A weighted (1/[nominal concentration]) least-squares linear regression of the type
Calibration curves for the analysis of NGR1, GRg1, and GRe in rabbit plasma and tissue.
Biosample | Calibration curves | Correlation coefficient |
Linear range ( |
---|---|---|---|
NGR1 | |||
Plasma |
|
0.9990 | 0.280–56.0 |
Heart |
|
0.9992 | |
Liver |
|
0.9990 | |
Brain |
|
0.9996 | |
Lung |
|
0.9992 | |
Kidney |
|
0.9996 | |
GRg1 | |||
Plasma |
|
0.9988 | 0.307–60.4 |
Heart |
|
0.9982 | |
Liver |
|
0.9986 | |
Brain |
|
0.9996 | |
Lung |
|
0.9990 | |
Kidney |
|
0.9990 | |
GRe | |||
Plasma |
|
0.9996 | 0.362–54.3 |
Heart |
|
0.9988 | |
Liver |
|
0.9992 | |
Brain |
|
0.9986 | |
Lung |
|
0.9988 | |
Kidney |
|
0.9990 |
The limit of detection (LOD) was estimated as the amount of NGR1, GRg1, and GRe, which caused a signal three times that of noise (
Data for intraday and interday precision and accuracy assessed by analyzing QC samples at different concentrations are presented in Table
The interday and intraday precision and accuracy of the method for the determination of NGR1, GRg1, and GRe (
Biosample | QC conc |
Intraday | Interday | Extraction recovery | |||
---|---|---|---|---|---|---|---|
Precision |
Accuracy |
Precision |
Accuracy |
Mean ± S.D. | R.S.D % | ||
NGR1 | |||||||
0.5 | 10.4 | 96.0 | 13.0 | 92.0 |
|
5.1 | |
Plasma | 5.0 | 6.7 | 102.0 | 11.1 | 94.0 |
|
3.8 |
35.0 | 4.2 | 97.4 | 5.4 | 103.7 |
|
7.9 | |
0.5 | 6.3 | 92.5 | 8.4 | 104.9 |
|
7.0 | |
Heart | 5.0 | 8.3 | 91.8 | 9.3 | 98.9 |
|
8.4 |
50.0 | 4.9 | 100.3 | 5.3 | 106.1 |
|
10.6 | |
0.5 | 8.7 | 98.1 | 10.6 | 95.8 |
|
8.4 | |
Liver | 5.0 | 7.5 | 91.4 | 8.3 | 96.6 |
|
4.9 |
50.0 | 7.1 | 99.6 | 7.8 | 105.6 |
|
7.4 | |
0.5 | 9.5 | 90.0 | 6.3 | 108.3 |
|
10.4 | |
Brain | 5.0 | 4.2 | 108.3 | 9.7 | 95.7 |
|
7.8 |
50.0 | 3.7 | 103.5 | 7.9 | 95.6 |
|
5.3 | |
0.5 | 7.4 | 94.2 | 13.1 | 103.7 |
|
8.5 | |
Lung | 5.0 | 12.1 | 98.6 | 4.8 | 105.5 |
|
8.6 |
50.0 | 6.7 | 105.8 | 10.2 | 95.4 |
|
10.1 | |
0.5 | 8.2 | 90.2 | 5.4 | 90.8 |
|
6.2 | |
Kidney | 5.0 | 11.1 | 91.4 | 3.8 | 98.4 |
|
4.0 |
50.0 | 5.9 | 90.5 | 7.8 | 91.3 |
|
5.6 | |
GRg1 | |||||||
0.4 | 14.3 | 105.1 | 13.5 | 92.5 |
|
4.5 | |
Plasma | 3.0 | 4.4 | 90.0 | 9.0 | 103.3 |
|
8.1 |
40.0 | 4.6 | 95.3 | 4.1 | 98.3 |
|
5.4 | |
0.4 | 10.2 | 97.4 | 6.4 | 96.3 |
|
10.2 | |
Heart | 3.0 | 4.4 | 93.9 | 7.5 | 94.7 |
|
7.8 |
40.0 | 6.2 | 101.3 | 10.4 | 104.6 |
|
8.2 | |
0.4 | 9.9 | 97.2 | 12.3 | 98.4 |
|
7.9 | |
Liver | 3.0 | 12.4 | 92.5 | 7.8 | 96.2 |
|
10.5 |
40.0 | 6.3 | 90.9 | 6.8 | 102.5 |
|
12.3 | |
0.4 | 8.9 | 108.9 | 13.2 | 90.4 |
|
6.5 | |
Brain | 3.0 | 6.1 | 96.3 | 8.4 | 94.3 |
|
5.7 |
40.0 | 7.3 | 101.8 | 9.3 | 103.1 |
|
8.3 | |
0.4 | 11.8 | 91.9 | 8.8 | 92.8 |
|
8.6 | |
Lung | 3.0 | 8.4 | 98.0 | 7.5 | 91.9 |
|
4.9 |
40.0 | 6.2 | 104.7 | 5.4 | 108.2 |
|
7.3 | |
0.4 | 5.4 | 92.8 | 10.2 | 98.7 |
|
10.5 | |
Kidney | 3.0 | 7.2 | 91.5 | 5.4 | 90.4 |
|
3.8 |
40.0 | 6.1 | 99.2 | 6.7 | 92.5 |
|
7.6 | |
GRe | |||||||
0.8 | 8.4 | 103.7 | 11.5 | 97.5 |
|
6.7 | |
Plasma | 8.0 | 6.6 | 92.5 | 6.2 | 107.5 |
|
7.9 |
48.0 | 4.2 | 104.7 | 3.8 | 102.9 |
|
7.8 | |
0.8 | 5.4 | 95.8 | 5.8 | 92.6 |
|
9.3 | |
Heart | 8.0 | 6.8 | 99.4 | 8.3 | 101.5 |
|
5.8 |
48.0 | 5.5 | 109.1 | 6.7 | 103.8 |
|
8.8 | |
0.8 | 9.1 | 96.8 | 10.8 | 98.3 |
|
10.5 | |
Liver | 8.0 | 6.8 | 94.5 | 9.6 | 96.1 |
|
6.6 |
48.0 | 9.1 | 96.8 | 8.4 | 98.0 |
|
7.3 | |
0.8 | 12.1 | 91.0 | 7.7 | 99.5 |
|
6.7 | |
brain | 8.0 | 8.2 | 109.5 | 13.2 | 104.3 |
|
9.3 |
48.0 | 7.3 | 104.8 | 9.4 | 93.9 |
|
6.8 | |
0.8 | 7.8 | 92.6 | 9.8 | 96.3 |
|
9.4 | |
Lung | 8.0 | 8.5 | 96.4 | 11.4 | 101.6 |
|
7.7 |
48.0 | 4.7 | 95.9 | 7.8 | 94.8 |
|
6.9 | |
0.8 | 6.1 | 91.8 | 8.5 | 92.8 |
|
8.1 | |
Kidney | 8.0 | 3.3 | 92.2 | 9.0 | 90.6 |
|
4.7 |
48.0 | 8.9 | 96.4 | 4.5 | 95.4 |
|
6.0 |
The extraction recovery analysis was conducted with NGR1, GRg1, and GRe spiked biosamples at three QC levels and calculated by comparing the NGR1, GRg1, and GRe peak areas in extracted biosamples with those found by direct injection of standard solutions at the same concentration. The mean recoveries of NGR1, GRg1, and GRe in plasma and tissue samples at three different concentrations were above 90.0% (Table
The stability studies were performed by evaluating small variations in three different conditions. The results were expressed as the percentage of initial content of NGR1, GRg1, and GRe in the freshly treated samples, suggesting that NGR1, GRg1, and GRe showed no significant change in plasma and tissue samples (Table
Stability of of NGR1, GRg1, and GRe in plasma samples and tissue homogenates of rabbits (
Biosample | QC conc ( |
Remaining (mean ± S.D.) | ||
---|---|---|---|---|
Short-term stability | Long-term stability | Freeze-thaw stability | ||
NGR1 | ||||
0.5 |
|
|
|
|
Plasma | 5.0 |
|
|
|
35.0 |
|
|
|
|
0.5 |
|
|
|
|
Heart | 5.0 |
|
|
|
50.0 |
|
|
|
|
0.5 |
|
|
|
|
Liver | 5.0 |
|
|
|
50.0 |
|
|
|
|
0.5 |
|
|
|
|
Brain | 5.0 |
|
|
|
50.0 |
|
|
|
|
0.5 |
|
|
|
|
Lung | 5.0 |
|
|
|
50.0 |
|
|
|
|
0.5 |
|
|
|
|
Kidney | 5.0 |
|
|
|
50.0 |
|
|
|
|
GRg1 | ||||
0.4 |
|
|
|
|
Plasma | 3.0 |
|
|
|
40.0 |
|
|
|
|
0.4 |
|
|
|
|
Heart | 3.0 |
|
|
|
40.0 |
|
|
|
|
0.4 |
|
|
|
|
Liver | 3.0 |
|
|
|
40.0 |
|
|
|
|
0.4 |
|
|
|
|
Brain | 3.0 |
|
|
|
40.0 |
|
|
|
|
0.4 |
|
|
|
|
Lung | 3.0 |
|
|
|
40.0 |
|
|
|
|
0.4 |
|
|
|
|
Kidney | 3.0 |
|
|
|
40.0 |
|
|
|
|
GRg1 | ||||
0.4 |
|
|
|
|
Plasma | 3.0 |
|
|
|
40.0 |
|
|
|
|
0.4 |
|
|
|
|
Heart | 3.0 |
|
|
|
40.0 |
|
|
|
|
0.4 |
|
|
|
|
Liver | 3.0 |
|
|
|
40.0 |
|
|
|
|
0.4 |
|
|
|
|
Brain | 3.0 |
|
|
|
40.0 |
|
|
|
|
0.4 |
|
|
|
|
Lung | 3.0 |
|
|
|
40.0 |
|
|
|
|
0.8 |
|
|
|
|
Kidney | 8.0 |
|
|
|
48.0 |
|
|
|
After oral administration of
The statistical parameters of NGR1, GRg1, and GRe after administration of
Parameters |
|
|
||||
---|---|---|---|---|---|---|
NGR1 | GRg1 | GRe | NGR1 | GRg1 | GRe | |
|
|
|
|
|
|
|
|
|
|
|
0.010 ± 0.003* | 0.010 ± 0.001 | 0.010 ± 0.001 |
|
|
|
|
28.4 ± 3.2** | 22.3 ± 3.1** | 25.2 ± 2.4** |
|
|
|
|
69.3 ± 5.2** | 69.3 ± 10.7 | 69.3 ± 15.2 |
V/F (L · kg−1) |
|
|
|
58.8 ± 6.9** | 35.9 ± 8.7* | 31.1 ± 6.9* |
CL/F (L · min−1 · kg−1) |
|
|
|
0.506 ± 0.027 | 0.119 ± 0.040 | 0.143 ± 0.054 |
|
|
|
|
306.3 ± 82.9** | 545.1 ± 51.7 | 525.1 ± 101.3 |
|
|
|
|
395.3 ± 101.4** | 1674.6 ± 148.2** | 1400.6 ± 251.9** |
|
|
|
|
0.009 ± 0.002* | 0.003 ± 0.00 | 0.005 ± 0.001 |
|
|
|
|
0.015 ± 0.005** | 0.025 ± 0.005** | 0.022 ± 0.005** |
|
|
|
|
0.011 ± 0.002 | 0.013 ± 0.001 | 0.011 ± 0.003 |
|
|
|
|
0.037 ± 0.005** | 0.051 ± 0.010* | 0.034 ± 0.003** |
|
|
|
|
1.62 ± 0.30 | 2.87 ± 0.34** | 3.04 ± 0.24** |
|
|
|
|
30.0 ± 8.0* | 30.0 ± 0.0 | 45.0 ± 13.4 |
|
|
|
|
18.8 ± 3.1** | 13.5 ± 4.6 | 20.3 ± 4.2** |
|
|
|
|
1.04 ± 0.21** | 1.25 ± 0.34** | 0.61 ± 0.47* |
Plasma concentration-time curves of NGR1 (a), GRg1 (b), and GRe (c) after administration of
It can be noted that the highest values of GRg1 were approximately the same as the values of GRe. This partly ascribed to the similar chemical properties of the two compounds. In addition, the increasing tendency of total distribution volume (
Combined with Borneol, the values of
In contrast to the pharmacokinetics of NGR1 in the
As listed in Table
Drug concentrations in rabbit tissues after administration of
Time ( |
Biosample | Concentration ( | |||||
---|---|---|---|---|---|---|---|
|
| ||||||
NGR1 | GRg1 | GRe | NGR1 | GRg1 | GRe | ||
0.5 | Heart | 3.90 ± 0.53 | 2.21 ± 0.76 | 1.65 ± 0.53 | 4.68 ± 0.21** | 22.65 ± 0.36** | 2.81 ± 0.74** |
Liver | 1.38 ± 0.54 | 8.48 ± 0.53 | 0.99 ± 0.33 | 8.24 ± 0.42** | 50.10 ± 1.95** | 3.60 ± 0.46** | |
Brain |
|
|
1.05 ± 0.42 | 4.02 ± 0.46** | 20.57 ± 1.36** | 1.80 ± 0.42* | |
Lung |
|
|
0.70 ± 0.41 | 5.79 ± 0.29** | 15.09 ± 3.24** | 2.48 ± 0.69** | |
Kidney |
|
|
1.63 ± 0.18 | 3.98 ± 0.12** | 27.54 ± 0.17** | 2.84 ± 0.53** | |
Plasma ( |
|
|
1.78 ± 0.11 | 1.62 ± 0.07 | 2.87 ± 0.06** | 2.68 ± 0.13** | |
| |||||||
1.0 | Heart |
|
|
2.11 ± 0.28 | 5.55 ± 0.31** | 27.03 ± 0.31** | 3.28 ± 0.43** |
Liver |
|
|
1.30 ± 0.25 | 9.29 ± 0.72** | 59.05 ± 3.74** | 4.77 ± 0.42** | |
Brain |
|
|
1.21 ± 0.28 | 5.58 ± 0.68** | 25.66 ± 2.69** | 2.76 ± 0.63** | |
Lung |
|
|
0.92 ± 0.13 | 9.96 ± 0.66** | 17.80 ± 1.25** | 2.99 ± 0.17** | |
Kidney |
|
|
1.53 ± 0.22 | 3.38 ± 0.34** | 16.93 ± 0.81** | 2.43 ± 0.29** | |
Plasma ( |
|
|
1.90 ± 0.15 | 1.46 ± 0.05 | 2.18 ± 0.09** | 2.42 ± 0.08** | |
| |||||||
3.0 | Heart |
|
|
1.14 ± 0.13 | 4.59 ± 0.52** | 19.07 ± 1.16** | 2.24 ± 0.54** |
Liver |
|
|
0.69 ± 0.11 | 6.87 ± 0.61** | 37.78 ± 3.43** | 2.63 ± 0.81** | |
Brain |
|
|
0.63 ± 0.12 | 3.69 ± 0.84** | 17.86 ± 2.60** | 1.65 ± 0.23** | |
Lung |
|
|
0.31 ± 0.44 | 4.56 ± 0.75** | 12.31 ± 1.46 | 1.53 ± 0.45** | |
Kidney |
|
|
1.15 ± 0.13 | 2.27 ± 0.28 | 10.16 ± 2.77** | 1.61 ± 0.45 | |
Plasma ( |
|
|
0.79 ± 0.02 | 0.49 ± 0.02 | 0.92 ± 0.01 | 0.87 ± 0.04 |
According to the classification method proposed by Yee [
Apparent permeability coefficients (
Compound |
|
|
|
---|---|---|---|
NGR1 | 0.64 ± 0.08 | 0.68 ± 0.12 | 1.06 |
GRg1 | 3.48 ± 0.42 | 3.64 ± 0.29 | 1.05 |
GRe | 5.46 ± 0.40 | 5.73 ± 0.37 | 1.05 |
NGR1 + Borneol | 1.87 ± 0.23** | 1.95 ± 0.34** | 1.04 |
GRg1 + Borneol | 9.05 ± 0.67** | 9.51 ± 0.62** | 1.05 |
GRe + Borneol | 12.67 ± 1.01** | 13.65 ± 1.59** | 1.08 |
Borneol is used as a “Guide drug” in traditional Chinese medicine, enhancing the expected functions of bioactive components from other herbs in the complex prescription through increasing bioavailability. Other research groups have found that Borneol could obviously loosen the intercellular tight junction, increase the number and volume of pinocytosis vesicles [
Effect of Borneol on TEER values of the Caco-2 cell monolayers. The Caco-2 cell monolayers were pretreated 2 h with 500
In summary, the present study showed that after combined oral administration to rabbits with
There is no conflict of interests to declare.
This work was supported by grants from the National Natural Science Foundation of China (Major International (Regional) Joint Research Project; Key Program, no. 81120108002; General Program, nos. 30930105 and 81071765), Program for Changjiang Scholars and Innovative Research Team in University of China (IRT1174), the Eleventh Five-Year National Science and Technology Support Program of China (no. 2008BAI51B01), and Natural Science Foundation of Shaanxi Province (nos. 2010JM4047 and 2012JZ4001), and The Education Department of Shaanxi Province (nos. 09JS086 and 11JK0661).