HPLC-MS/MS Analysis of Aconiti Lateralis Radix Praeparata and Its Combination with Red Ginseng Effect on Rat CYP450 Activities Using the Cocktail Approach

Red ginseng is often combined with Aconiti Lateralis Radix Praeparata to reduce alkaloids-related toxicity of the latter. Such herb-pairing also results in better therapeutic effect in heart failure, as compared to the singular use of either herb. The purpose of this study was to investigate the effect of Aconiti Lateralis Radix Praeparata and its combination with red ginseng on the activities of CYP450 enzymes in rats. A sensitive and reliable HPLC-MS/MS method was established and validated for the simultaneous determination of eight probe drugs, phenacetin (CYP1A2), tolbutamide (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), dapsone (CYP3A4), 7-hydroxycoumarin (CYP2A6), bupropion (CYP2B6), and amodiaquine (CYP2C8), in rat plasma using diazepam as internal standard (IS). The chromatographic separation was performed on a Waters XBridge™ C18 column (2.1 mm × 100 mm, 3.5 μm) using a gradient elution with the mobile phase consisting of acetonitrile and water (containing 0.1% formic acid) at a flow rate of 0.3 mL/min. The method was successfully applied in evaluating the effect of Aconiti Lateralis Radix Praeparata and red ginseng on the activities of CYP450 enzymes. The pharmacokinetic results of the eight probe drugs suggested that Aconiti Lateralis Radix Praeparata may inhibit the activity of CYP2A6, CYP2C19, CYP2B6, CYP1A2, CYP3A4, and CYP2C9 enzymes in rats. Comparison between the two groups, Aconiti Lateralis Radix Praeparata combined with red ginseng and Aconiti Lateralis Radix Praeparata, indicated that red ginseng may inhibit the activity of CYP2D6 and CYP2B6 enzymes while inducing the activity of CYP1A2, CYP3A4, and CYP2C9 enzymes.


Introduction
Aconiti Lateralis Radix Praeparata ("Fuzi" in Chinese), recorded in Chinese Pharmacopoeia (2015 edition), is the lateral root of Aconitum carmichaelii Debx., belonging to family Ranunculaceae. e major chemical components of Aconiti Lateralis Radix Praeparata are alkaloids including diester diterpenoid alkaloids, monoester diterpenoid alkaloids, and lipid alkaloids. Aconiti Lateralis Radix Praeparata has multiple pharmacological properties, including antiarrhythmia, anti-inflammatory, antitumor, and antiaging properties, but it has very narrow therapeutic range due to its severe toxicity [1][2][3][4]. Clinically, Aconiti Lateralis Radix Praeparata is commonly prescribed with red ginseng ("Hongshen" in Chinese, derived from the steamed root and rhizome of Panax ginseng C. A. Mey.) for treating heart failure [5]. Previous studies have found that the combination of red ginseng and Aconiti Lateralis Radix Praeparata is able to reduce the contents of diester diterpenoid alkaloids (hypaconitine and deoxyaconitine) and increase the contents of monoester diterpenoid alkaloids (benzoylmesaconine and benzoylhypaconine) in Aconiti Lateralis Radix Praeparata, thus strengthening the therapeutic effect of Aconiti Lateralis Radix Praeparata while decreasing its toxicity [6,7]. However, the mechanism of action of Aconiti Lateralis Radix Praeparata combined with red ginseng is still unclear in vivo.
CYP450 family is the most important drug-metabolizing system, playing a vital role in the metabolism of endogenous and exogenous compounds [8,9]. It is responsible for the metabolism of over 70-80% of the rate-limiting phase I metabolism of drugs [10]. More than 90% of clinical drugs are metabolized by the CYP450 enzymes, including CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP2A6, CYP2B6, and CYP2C8 [11]. Clinically, induction or inhibition of the CYP450 enzyme activities has been regarded as the foremost contributor to assess drug-drug interactions (DDIs) [12]. Evaluation of the effect of drugs, especially the combination of traditional Chinese medicine, on CYP450 enzyme activities is increasingly important, since it can explain the mechanism of action of DDIs or predict the potential side effects [13,14]. Recent studies identified that CYP450 enzymes such as CYP2D6 and CYP3A4 were involved in the metabolism of Aconiti Lateralis Radix Praeparata-red ginseng herbal formulation. Compounds such as Ginsenosides Rc, Rf, and Rb2 induced CYP3A4 metabolic enzymes [15,16], while diester alkaloids of Aconiti Lateralis Radix Praeparata were identified as substrates of CYP3A4 metabolic enzymes [17]. However, several Ginsenoside components or in vitro studies have not been able to fully elucidate the effects of Aconiti Lateralis Radix Praeparata and its combination with red ginseng on CYP450 enzymes.
Cocktail approach has become one of the basic analytical tools to assess the impact of DDIs on CYP450 enzymes in vivo. is method not only provides information of multiple CYP450 enzymes activities in a single experiment but also minimizes the intersubject and intrasubject variability [18][19][20]. Compounds specifically catalyzed by each CYP450 enzyme, regarded as probe drugs, have been widely used to evaluate the activity of CYP450 enzymes in this method [18,21]. CYP450 enzymes inhibition or induction by one drug could lead to the increase or reduction in plasma concentrations of another drug, thereby enhancing or attenuating the therapeutic effect [22]. In this study, a rapid and sensitive LC-MS/MS method was established for the quantitation of specific probe drugs in rat plasma to evaluate the influence of Aconiti Lateralis Radix Praeparata and its combination with red ginseng on CYP450 enzymes. e results would be significant in explaining the mechanism of toxic-reduction and therapeutic-enhancing effects of Aconiti Lateralis Radix Praeparata and red ginseng, which would provide a theoretical basis for rational usage of red ginseng and Aconiti Lateralis Radix Praeparata in clinical.

Experimental
2.1. Chemicals, Reagents, and Materials. Acetonitrile (chromatographic purity) and methanol (chromatographic purity) were purchased from Merck Co., Ltd. Formic acid (chromatographic purity) was obtained from ROE Co., Ltd. Ultrapure water was prepared with a Milli-Q water purification system (Millipore, Milford, MA, USA). Omeprazole, dextromethorphan, bupropion, 7-hydroxycoumarin, and amodiaquine were purchased from the China National Institutes for Food and Drug Control. Phenacetin, tolbutamide, and diazepam were obtained from Tianjin Vientiane Hengyuan Technology Co., Ltd. Dapsone was purchased from J&K Scientific Ltd. e extracts of red ginseng and Aconiti Lateralis Radix Praeparata were made in the laboratory, both of which were extracted with 70% ethanol. e extraction rates of red ginseng and Aconiti Lateralis Radix Praeparata were 43.36% and 15.38%, respectively.

Chromatographic and Mass Spectrometry Conditions.
e HPLC-MS/MS system consists of an Agilent 1200 highperformance liquid chromatography coupled with an Agilent 6430 series triple quadrupole mass spectrometer with an electrospray ionization (ESI) source. e chromatographic separation was performed on a Waters XBridge ™ C18 column (2.1 × 100 mm, 3.5 μm) maintained at 30°C. Mobile phases consisting of 0.1% formic acid in water (A) and acetonitrile (B) were used in the following gradient elution method: 0-2 min, 10%-30% B; 2-7 min, 30%-80% B; and 7-10 min, 80%-80% B. e flow rate was 0.3 mL/min, and the injection volume was 5 μL. All data were analyzed by Mass Hunter workstation software (Agilent Technologies, USA). e mass spectrometer was carried out in positive ionization multiple reaction monitoring (MRM) mode. e optimum MS values were maintained as follows: nebulizing gas pressure, 25 psi; drying gas (N 2 ) flow rate, 11 L/min; and capillary temperature, 350°C. e precursor-product ion pairs used for the MRM detection and MS parameters are shown in Table 1.

Preparation of Aconiti Lateralis Radix Praeparata and Red Ginseng Solution.
e dosage of Aconiti Lateralis Radix Praeparata extract was 0.415 g/kg. According to the raw herb combination ratios of Aconiti Lateralis Radix Praeparata and red ginseng (1 : 2), the dosage of red ginseng extract was 2.34 g/kg. Appropriate amounts of Aconiti Lateralis Radix Praeparata and red ginseng were weighed, prepared with 0.5% CMC-Na suspension solution, and stored at 4°C.
Quality control (QC) samples at three concentrations (low, medium, and high concentration) were made up of appropriate mixed standard solutions with blank plasma and IS solutions as calibration standard solutions to meet the required concentrations. All the solutions were stored at 4°C.

Sample Preparation.
100 μL plasma sample, 20 μL IS (diazepam, 1000 ng/mL), and 20 μL methanol were added to a centrifuge tube and mixed by vortexing. Liquid-liquid extraction was performed by adding 1 mL ethyl acetate to the centrifuge tube. e mixture was then vortexed for 3 min and centrifuged at 14,000 rpm for 10 min. e supernatant was transferred into a new tube and evaporated to dryness under a nitrogen stream. e residue was reconstituted with 100 μL methanol and vortexed for 3 min, followed by centrifugation at 14,000 rpm for 10 min. 5 μL supernatant was injected into the LC-MS/MS system for analysis.

Method Validation.
e method was validated with respect to specificity, linearity, accuracy, precision, extraction recovery, matrix effect, and stability according to bioanalytical method validation.

Specificity.
e specificity of the method was investigated by comparing chromatograms of blank plasma samples from six different rats, blank plasma spiked with the eight probe drugs and IS, and a rat plasma sample after the tail vein administration of cocktail probes.

Linearity and LLOQ.
Calibration curves were achieved using a series of calibration standard samples and constructed by plotting the peak area ratio analyte to IS (y) versus the concentration of analyte in spiked plasma samples (x), using 1/x 2 as a weighing factor. e lower limit of quantification (LLOQ) was determined at a signal-to-noise ratio of about 10 by analyzing the standard plasma samples.

Precision and Accuracy.
e precision and accuracy were evaluated by determination of QC samples in six replicates at low, medium, and high concentration levels.
e precision and accuracy were analyzed for 3 consecutive days with the standard calibration curve. Intra-and interday precisions were expressed as the relative standard deviation (RSD), while the accuracy was determined by the relative error (RE).

Extraction Recovery and Matrix Effect.
e extraction recovery and matrix effect of eight analytes were calculated at three concentration levels, each in six replicates. e extraction recovery was calculated using the ratio of peak area of the extracted samples and that of postextraction spiked samples. e matrix effect was obtained by comparing the peak areas of the postextraction spiked samples versus standard solutions.

Stability.
e stability of eight probe drugs was analyzed in six replicates at three concentration levels. e QC samples were prepared for 4 h at room temperature, three freeze-thaw cycles, 12 h in autosampler, and 14 days at − 70°C.

Pharmacokinetic Study.
Male Sprague-Dawley rats (190-230 g) were obtained from Beijing HFK Experimental Animal Technology Co., Ltd. e rats were fed with standard diet and water in a stable environment, with temperatures between 23 and 26°C and relative humidity of 40-60%. After a week of acclimation, the animals were fasted 12 h before the PK experiment and allowed free access to water during the experiment. 18 rats were randomly divided into three groups. e rats were given 10 mL/kg cocktail solution (2 mg/kg of 7-hydroxycoumarin; 0.2 mg/kg of tolbutamide; 1 mg/kg of omeprazole, dextromethorphan, and bupropion; 0.5 mg/kg of dapsone and phenacetin; and 3 mg/kg of amodiaquine) through the tail vein in group A. e rats were administered 0.415 g/kg Aconiti Lateralis Radix Praeparata extract by oral administration for 7 consecutive days and then dosed 10 mL/kg cocktail solution through the tail vein on the eighth day in group B. e rats of group C were administered 0.415 g/kg Aconiti Lateralis Radix Praeparata extract and 2.34 g/kg red ginseng extract by oral administration for 7 consecutive days and dosed 10 mL/kg cocktail solution through the tail vein on the eighth day. Blood

Optimization of HPLC-MS/MS Conditions.
Acetonitrile was selected as the mobile phase as it has strong elution ability. e responses of eight probe drugs and IS were improved after adding 0.1% formic acid to water. e analytes have good retention time, peak symmetry, and appropriate ionization in the mobile phase with gradient elution. e eight probe drugs were monitored in positive and negative ion modes, and it was discovered that ions in positive mode had more stable and stronger MS signal than in negative mode. e transitions m/z 346. e representative chromatograms of a blank plasma (A), a rat plasma sample after the tail vein administration of cocktail probes at 10 mL/kg (B), and blank plasma sample spiked with probe drugs and IS (C) are shown        Evidence-Based Complementary and Alternative Medicine in Figure 1. As shown in Figure 1, no significant interfering peaks from endogenous substance were observed at the corresponding analytes retention time.

Linearity and LLOQ.
e curves were linear (r > 0.991) in the calibration ranges of each probe drug in rat plasma. e regression equations, linear ranges, correlation coefficients, and LLOQ are displayed in Table 2. e results indicated that the method was linear and sensitive. Table 3, the intra-and interday precisions at three different concentration levels of eight probe drugs were 13.0% or less, while the accuracy ranged from − 14.0% to 14.4%. e results of the precision and accuracy met the criteria for the analysis of biological samples.

Extraction Recovery and Matrix Effect.
e mean extraction recoveries in rat plasma ranged from 85.0% to 109.4% for the eight probe drugs at three concentration levels. e mean matrix effects of the eight analytes were between 81.5% and 105.6%. e extraction recovery and matrix effect data are summarized in Table 4.

Stability.
e results of stability are displayed in Table 5. Eight probe drugs were stable with RSD range of 0.4%-14.3% under various storage conditions.

Effects of Aconiti Lateralis Radix Praeparata and Its Combination with Red Ginseng on CYP450 Activities in Rats.
e method was applied to pharmacokinetic study of eight probe drugs in rats. e mean plasma concentration-time curves of the eight cocktail probe drugs are shown in Figure 2, and the main pharmacokinetic parameters of eight probe drugs are summarized in Table 6.

Effects of Aconiti Lateralis Radix Praeparata on Eight
Probe Drugs in Rats. Pharmacokinetic profiles of eight probe drugs after Aconiti Lateralis Radix Praeparata treatment were used to estimate the activity of CYP450 enzymes in rats. As compared to group A, C max , AUC (0-24) , and AUC (0-∞) of 7hydroxycoumarin, omeprazole, bupropion, phenacetin, dapsone, and tolbutamide in group B were increased, and MRT (0-24) of phenacetin, dapsone, and tolbutamide were significantly added (P < 0.05 or P < 0.01). e results suggested that Aconiti Lateralis Radix Praeparata may inhibit the activity of CYP2A6, CYP2C19, CYP2B6, CYP1A2, CYP3A4, and CYP2C9 enzymes in rats. e pharmacokinetic parameters of dextromethorphan and amodiaquine in group B had no significant differences when compared to group A, implying that Aconiti Lateralis Radix Praeparata showed little alteration on CYP2D6 and CYP2C8 activity in vivo.

Effects of Aconiti Lateralis Radix Praeparata Combination with Red Ginseng on Eight Probe Drugs in Rats.
Alteration in CYP450 enzymes activity caused by red ginseng was estimated by comparing the pharmacokinetic results of eight probe drugs in rats by different treatment methods. Compared with group B, C max , AUC  , and AUC (0-∞) of dextromethorphan and bupropion in group C were significantly increased (P < 0.01), while the half-life (t 1/2 ) of dextromethorphan and bupropion was markedly reduced (P < 0.01). e results showed that metabolism of dextromethorphan and bupropion in group C was slowed. But C max , AUC (0-24) , and AUC (0-∞) of phenacetin, dapsone, and tolbutamide were  significantly decreased (P < 0.05 or P < 0.01), indicating that red ginseng may enhance the metabolism of phenacetin, dapsone, and tolbutamide. us, these pharmacokinetic parameters suggested that red ginseng may inhibit the activity of CYP2D6 and CYP2B6 enzymes but induce the activity of CYP1A2, CYP3A4, and CYP2C9 enzymes in rats. e toxicity of Aconiti Lateralis Radix Praeparata is mainly attributed to its diester diterpenoid aconitines such as aconitine, hypaconitine, and mesaconitine [23,24]. According to reports, diester diterpenoid aconitines of Aconiti Lateralis Radix Praeparata are substrates of CYP3A4 metabolic enzymes [10]. Further pharmacokinetic studies were carried out on Aconiti Lateralis Radix Praeparata and its combination with red ginseng (Figure 3). C max and AUC (0-∞) of aconitine, hypaconitine, and mesaconitine of Aconiti Lateralis Radix Praeparatared ginseng were decreased as compared to those of Aconiti Lateralis Radix Praeparata administration. erefore, red ginseng may accelerate the metabolism of toxic components in Aconiti Lateralis Radix Praeparata by inducing the activity of CYP3A4 enzymes. is experiment provides a theoretical basis for explaining the interaction mechanism of Aconiti Lateralis Radix Praeparata combined with red ginseng, which would provide reference for rational usage of red ginseng and Aconiti Lateralis Radix Praeparata in clinic.

Conclusion
In this study, a sensitive and rapid LC-MS/MS method was developed and validated for the simultaneous determination of eight probe drugs in rat plasma to evaluate the effect of Aconiti Lateralis Radix Praeparata and its combination with red ginseng on rat CYP450 enzymes. e results indicated that Aconiti Lateralis Radix Praeparata may inhibit the activity of CYP2A6, CYP2C19, CYP2B6, CYP1A2, CYP3A4, and CYP2C9 in rats. Comparison between the two groups of Aconiti Lateralis Radix Praeparata with or without red ginseng showed that red ginseng may inhibit the activity of CYP2D6 and CYP2B6 while inducing the activity of CYP1A2, CYP3A4, and CYP2C9 enzymes.

Evidence-Based Complementary and Alternative Medicine
Data Availability e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest.