Banha-sasim-tang (BST), which consists of seven different herbs, is one of the most popular herbal formulae for treating gastrointestinal disorders in Eastern Asia. The commonly used herbal medicine is often co-administered with other therapeutic drugs, which raises the possibility of herb–drug interactions and may modify the clinical safety profile of therapeutic drugs.
We investigated the potential herb–drug interactions between BST extract and midazolam (MDZ) in mice. The area under the plasma concentration-time curve (AUC) of MDZ and 1ʹ-hydroxymidazolam (1ʹ-OH-MDZ) was evaluated for both oral and intraperitoneal administration of MDZ, following oral administration of BST (0.5 and 1 g/kg).
It was found that the AUC of MDZ and 1ʹ-OH-MDZ was lower in case of oral administration of MDZ. Administration of BST extract was not associated with hepatic cytochrome P450 activity. BST extract induced a strong reduction in pH and it has been reported that oral mucosal absorption of MDZ is lower at low pH. The decreased absorption rate of MDZ might be caused by the ingredients of BST and may not be related to other factors such as increased excretion of MDZ by P-glycoprotein.
The altered pharmacokinetics of midazolam caused by co-administration with BST
Herbal medicines are commonly used to treat various diseases worldwide and are often co-administered with therapeutic drugs. However, the co-administration of herbs and drugs raises the potential of herb–drug interactions, which may affect the clinical safety of therapeutic drugs. Herb–drug interactions can be predicted by evaluating pharmacokinetic parameters, including absorption, metabolism, distribution, and excretion of drugs. Several drugs or herbs may affect the activity of drug-metabolizing enzymes, especially cytochrome P450 (CYP), resulting in herb–drug or drug–drug interactions [
Commonly known as Banha-sasim-tang (BST) in Korea, or Ban-xia-xie-xin tang in China, the herb under study is one of the most popular herbal formulae mentioned in old herbal prescription literature [
To prevent or minimize adverse herb–drug interactions, herbal medicine that interacts with drugs should be investigated in both
BST solid extract was obtained from Hanzung Pharmaceutical (Daejeon, Korea) and prepared in accordance with the Korean Pharmacopoeia (KP), 10th edition. The preparation contained the following ingredients: water extract of
Specific pathogen-free, 5-week-old male ICR mice (24 to 26 g) were obtained from Orient Bio (Seongnam, Korea) and acclimated for at least seven days before the experiment. Upon arrival, the animals were randomly housed in cages, with four or five per cage. The animal quarters were strictly maintained at 23 ± 3°C and 50 ± 10% relative humidity with a 12 hours light/dark cycle (intensity: 150–300 Lux). All animal procedures were performed in accordance with the Society of Toxicology guidelines of 1989. The study was approved by the institutional review board of the Kyungpook National University (2015-0099).
A total of 18 male ICR mice (30 ± 2 g) were randomly divided into a vehicle-treated, and two MDZ-treated groups, which also received BST extract dissolved in saline. In the first group, MDZ (2.0 mg/kg) was orally administered to nine mice after 5 minutes of oral administration of BST extract (0, 0.5 or 1.0 g/kg, n = 3). In the second group, nine mice were intraperitoneally treated with MDZ (2.0 mg/kg) after 5 minutes of oral administration of BST extract (0, 0.5 or 1.0 g/kg, n = 3). After the mice received MDZ, blood samples were collected from the tail vein into heparinized capillary tubes at 0.08-, 0.167-, 0.25-, 0.5-, 1-, and 2 hour time-points. The collected blood was immediately centrifuged at 4000 g for 10 minutes and 10 μL of plasma was obtained for each sample. The samples were stored at −20°C until analysis.
Plasma samples (10 μL) were added to 90 μL of acetonitrile (ACN) with 0.1% formic acid and 5 μM reserpine solution (internal standard [IS], 97.5:2.5, v/v) was added. The solution was mixed and centrifuged at 13000 rpm for 10 minutes at 4°C, and 90 μL of the supernatant was obtained. The supernatants were transferred to autosampler vials, and 5 μL aliquots were injected into the LC system.
All measurements were performed using an LC-MS/MS system in the selective reaction monitoring (SRM) mode. A Triple Stage Quadrupole Vantage mass spectrometer an HESI-II spray source coupled to an Accela™ LC system (Thermo Fisher Scientific, Waltham, MA, USA) was employed. The vaporizer and capillary temperatures were set to 150°C and 300°C, respectively. Electrospray ionization was performed in the positive mode at a spray voltage of 3500 V. Nitrogen was used as the sheath and auxiliary gas, and was set to 45 and 20 (arbitrary units), respectively. Data were analyzed using the Xcalibur software (Thermo Fisher Scientific). An ACE® 5C18 column (5 μm, 50 mm × 2.1 mm, Advanced Chromatography Technologies, Scotland, UK) and a guard C18 column (2 μm, 2.1 mm ID, Phenomenex, Torrance, CA, USA) were employed for LC separation. The mobile phase consisted of ACN with 0.1% FA (mobile phase A) and water (mobile phase B) at a flow rate of 220 μL/min. The gradient was as follows: 0 minute 5% A, 0.5 minutes 5% A, 1.5 minutes 95% A, 3.0 minutes 95% A, 3.5 minutes 5% A, 5.0 minutes 5% A. Ions monitored in the SRM mode were m/z 326.0→291.0 for MDZ, 342.0→203.0 for 1’-OH-MDZ m/z, and 609.4→174.1 for the IS, respectively, at an SRM collision energy of 29 eV for MDZ, 15 eV for 1ʹ-OH-MDZ and 42 eV for IS. Data procurement was controlled using the Xcalibur software (Thermo Fisher Scientific).
The calibration curves of MDZ and 1′-OH-MDZ ranged from 0.05 to 2.0 μg/mL, respectively. Calibration curves were constructed by plotting the peak-area ratios of analyte or IS vs. the concentrations of MDZ and 1’-OH-MDZ in mouse plasma. The equations for the calibration curves of MDZ and 1ʹ-OHMDZ were y = 25114 x–13.85 (r2 = 0.999) and y = 19380x – 9.12 (r2= 0.998), respectively. The lower limits of quantification values of MDZ and 1ʹ-OH-MDZ were 50 and 50 ng/mL, respectively. The quality control samples of the methodology were evaluated by analyzing five replicates of mouse plasma spiked with known concentrations of MDZ (0.05, 0.5, 1.0, and 2.0 μg/mL) or 1’-OH-MDZ (0.05, 0.5, 1.0, and 2.0 μg/mL) (
The pH of BST extract was measured using a Metter Toledo S220 SevenCompact™ pH/Ion pH meter (Metter-Toledo International Inc., Columbus, OH, USA). The pH of each concentration of BST extract in the mixture was measured immediately after it dissolved.
A non-compartmental model was used to calculate the pharmacokinetic parameters, using WinNonlin version 2.1 (Scientific Consulting Inc., Cary, NC, USA), and includes maximum plasma concentration (Cmax), time to reach maximum plasma concentration (Tmax), area under the plasma concentration-time curve (AUC), and half-life.
The mean value ± standard error was determined for each treatment group of a given experiment. Dunnett’s
To investigate the potential herb–drug interactions observed during co-administration of BST with MDZ, two different administration methods were evaluated (
Mean plasma concentration–time profiles of MDZ and 1′-OH-MDZ were obtained (
ICR male mice were simultaneously treated with MDZ (2.0 mg/kg, intraperitoneal [IP]) following oral administration of BST (0.5 or 1.0 g/kg), to investigate the inhibitory effect of BST on CYP3A4 activity. The concentration-time profiles of MDZ and 1′-OH-MDZ in plasma are shown in
Our study showed that the pharmacokinetic parameters of MDZ and 1′-OH-MDZ decreased following oral co-administration with BST, but not in case of IP administration. When MDZ was orally administrated, the plasma concentration of MDZ was regulated by two mechanisms: absorption rate and metabolic stability in the liver. The plasma concentration of MDZ was found to be mainly affected by a mechanism involving metabolic conversion of MDZ in the liver. The plasma concentration of MDZ and its metabolites was not changed by BST IP co-administration, which indicates that BST does not have a strong inhibitory action on CYP activities in the liver. Moreover, a decrease in plasma concentration of MDZ and 1’-OH-MDZ is observed following oral co-administration with BST. The findings therefore indicate that BST affects the absorption of MDZ, but not its metabolism.
The reduction in plasma concentration of MDZ and 1′-OH-MDZ in animals co-administered with MDZ and BST (1.0 g/kg, PO) indicates that the absorption of MDZ might be inhibited by BST, and that this inhibition might not be associated with metabolic activity or CYP3A4 enzyme. The reduced absorption of MDZ may be attributed to several phenomena. One is the formation of insoluble particles from the components of BST extract interacting with MDZ at physiological pH conditions (pH 1-2 in the stomach, or pH 5-6 in the intestines). However, the concentrations of MDZ in the supernatants of the BST-MDZ mixture in artificial gastric juice or intestinal juice was not significantly altered (
Another hypothesis is that the reduced pH induced by BST extract in the stomach or intestines affects the oral mucosal absorption of MDZ, since it has been reported that MDZ absorption is strongly inhibited under low pH conditions [
The other possible mechanism involved in the phenomena under investigation is that the ingredients in BST inhibits the absorption of MDZ in the stomach or intestine. The complex ingredients in BST might compete with the absorption of MDZ. Since MDZ is not a substrate of P-glycoprotein (P-gp), the reduced plasma concentration cannot be related to increased excretion by P-gp [
Here, we investigated the herb–drug interactions between BST extract and MDZ
This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Export Promotion Technology Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant no. 316017-3).
The authors have no conflicts of interest associated with material presented in this paper.
The accuracy and RSD of quality control samples of MDZ and 1’-OH MDZ
Effects of co-administration of BST extract on the pharmacokinetic parameters of caffeine and paraxanthine
The concentration of MDZ in the supernatant of artificial gastric juice (A) and intestinal juice (B) with MDZ and BST. The data shown are the means of duplicates. MDZ, midazolam; BST, Banha-sasim-tang.
Experimental design. (A) Male ICR mice were orally administered MDZ (2.0 mg/kg) following administration of BST extract (0, 0.5, or 1.0 g/kg) or vehicle (saline) by oral gavage. (B) Male ICR mice were intraperitoneally treated with MDZ (2.0 mg/kg) following administration of BST extract (0, 0.5, or 1.0 g/kg) or vehicle (saline) by oral gavage. The blood samples were obtained and the concentrations of MDZ and 1ʹ-OH-MDZ (metabolite) were determined. MDZ, midazolam; BST, Banha-sasim-tang; 1ʹ-OH-MDZ, 1ʹ-hydroxymidazolam.
Mean plasma concentration-time profiles of MDZ and 1ʹ-OHMDZ in mice simultaneously treated with BST (0, 0.5, or 1.0 g/kg, PO) and MDZ (2.0 mg/kg, PO). Data are presented as mean±standard error (n=3). MDZ, midazolam; 1ʹ-OH-MDZ, 1ʹ-hydroxymidazolam; BST, Banha-sasimtang; PO, peroral.
Mean plasma concentration-time profiles of MDZ and 1ʹ-OH-MDZ in mice simultaneously treated with BST (0, 0.5, or 1.0 g/kg, PO) and MDZ (2.0 mg/kg, IP). Data are presented as mean ± standard error (n=3). MDZ, midazolam; 1ʹ-OH-MDZ, 1ʹ-hydroxymidazolam; BST, Banha-sasimtang; PO, peroral; IP, intraperitoneal.
PH values of the Banha-sasim-tang (BST) extract mixtures (0–100 mg/mL).
Effects of co-administration of BST extract with MDZ on the pharmacokinetic parameters of MDZ and 1ʹ-OH-MDZ (n=3)
Parameters | BST extract (g/kg) | Oral administration of MDZ |
Intraperitoneal administration of MDZ |
||||||
---|---|---|---|---|---|---|---|---|---|
AUC (mg-min/mL) | Cmax (mg/mL) | Half-life (min) | Tmax (min) | AUC (mg-min/mL) | Cmax (mg/mL) | Half-life (min) | Tmax (min) | ||
MDZ | 0.0 | 45.7±2.9 | 0.7±0.1 | 167.3±116.8 | 5.0±0.0 | 234.5±9.5 | 4.5±0.1 | 23.1±1.5 | 11.7±1.7 |
0.5 | 33.9±6.5 | 0.7±0.1 | 59.3±15.3 | 8.3±3.3 | 220.1±26.2 | 4.5±0.5 | 25.0±3.1 | 10.0±0.0 | |
1.0 | 16.9±4.1 |
0.3±0.1 | 79.1±1.2 | 26.7±16.9 | 197.9±7.8 | 3.9±0.2 | 23.1±0.8 | 9.0±1.3 | |
1'-OH MDZ | 0.0 | 3540.8±312.7 | 43.1±8.5 | 55.3±0.0 | 50.0±10.0 | 18.9±0.8 | 0.2±0.01 | 159.8±11.2 | 20.0±5.0 |
0.5 | 3189.7±907.0 | 42.9±12.6 | 71.8±12.3 | 16.7±7.3 | 18.2±0.3 | 0.1±0.01 | 166.0±6.1 | 13.8±1.3 | |
1.0 | 1901.0±194.2 |
21.4±2.5 | 101.1±1.7 | 31.7±15.9 | 18.2±0.4 | 0.1±0.0 | 169.0±11.3 | 21.0±3.8 |
Data are presents as mean±standard errors.
BST, Banha-sasim-tang; MDZ, midazolam; 1ʹ-OH-MDZ, 1ʹ-hydroxymidazolam; AUC, area under the plasma concentration-time curve; Cmax, maximum plasma concentration; Tmax, maximum plasma concentration