A High-Performance Liquid Chromatography with Photodiode Array Detection Method for Simultaneous Determination of Three Compounds Isolated from Wikstroemia ganpi: Assessment of the Effects on Cytochrome P450-Mediated Metabolism In Vitro and In Vivo

In natural products, the content and quality of the marker components differ depending on the part, production area, collection period, and extraction method; therefore, a standardized analysis method is required to obtain consistent results. This study developed a simultaneous analysis method for three marker components (7-methoxylutolin-5-O-glucoseide, pilloin 5-O-β-d-glucopyranoside, rutarensin) isolated and purified from Wikstroemia ganpi (W. ganpi). Simultaneous analysis was performed using high-performance liquid chromatography with photodiode array detection (HPLC-PDA) method that was validated according to the International Council for Harmonisation (ICH) guidelines. The developed analytical method exhibited linearity (r2 > 0.999), detection limits (0.72–3.34 μg/mL), and quantification limits (2.19–10.22 μg/mL). The relative standard deviation (RSD) value of intra- and inter-day precisions was less than 1.68%, and analyte recoveries (93.42–117.55%; RSD < 1.86%) were validated according to the analytical procedures, and all parameters were within the allowable range. Quantitative analysis of the three marker components from W. ganpi MeOH extract (WGM) showed 7-methoxylutolin-5-O-glucoseide with the highest content (51.81 mg/g). The inhibitory effects of WGM on cytochrome P450 (CYP) substrate drugs were further investigated. The in vitro study revealed that WGM inhibited the CYP3A-mediated metabolism of buspirone and that 7-methoxylutolin-5-O-glucoseide and pilloin 5-O-β-d-glucopyranoside inhibited the metabolism of buspirone with IC50 values of 2.73 and 18.7 μM, respectively. However, a single oral dose of WGM did not have significant effects on the pharmacokinetics of buspirone in rats, suggesting that WGM cannot function as an inhibitor of CYP3A-mediated metabolism in vivo.

Phenolic compounds, which are secondary plant metabolites, are categorized according to their chemical structure into simple phenols, phenolic acids, flavonoids, tannins, and coumarins [13].They are known to have antioxidant, antibacterial, and anti-allergic properties and to prevent hyperlipidemia [14].Flavonoids are phenolic compounds composed of two benzene and heterocyclic rings, which are known to inhibit the rate of oxidation and free radical formation via intracellular lipoxygenase activity [15].It has also been reported that cardiovascular diseases can be improved with their use, and there are also anti-cancer, antioxidant, and anti-inflammatory effects of flavonoids [16].Another phenolic compound, coumarin, is a heterocyclic compound belonging to the class of benzopyrones [17].Compounds with a coumarin skeleton have anti-inflammatory effects and are reported to be inhibitory in α-chymotrypsin and human leukocyte diastase [18].Natural products contain various substances other than pharmacological substances, and their composition ratios vary depending on habitat, climate, and site [19].Therefore, it is necessary to standardize the quality, purity, and reliability of analyte targets.Nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) are primarily used for the qualitative analysis of chemicals.NMR spectroscopy is less sensitive than MS spectroscopy but is more effective in identifying and quantifying metabolites in tissue extracts [20].The chemical profile can be used to analyze substances using chromatographic fingerprints such as high-performance liquid chromatography (HPLC) [21], gas chromatography (GC) [22], and capillary electrophoresis (CE) [23], and spectral fingerprints such as ultraviolet (UV), infrared (IR), and mass spectrometry (MS) [24,25].In particular, HPLC equipped with photodiode array (PDA) detectors has the advantage of being able to use a variety of solvents and is the most commonly used liquid chromatography method for material analysis.It is also a certified method for the overall quality assessment of drugs [26].
The analytical methods used for natural products should be validated (quality, safety, efficacy, etc.), and the International Council for Harmonisation (ICH) guidelines are appropriate for this [27].The ICH guidelines discuss the characteristics that should be considered when validating analytical procedures and are used to demonstrate the suitability of a developed analytical procedure for its intended purpose.Several countries refer to ICH Q2(R1) guidelines [28] because the statistical approach used in the validation of methods maximizes the efficiency of a validation test.The data presented for the optimal design of the test methods were as follows: specificity, linearity, range, accuracy, precision, and recovery.Due to insufficient phytochemical studies and data on marker components, standardization of the method is important for obtaining accurate and reliable results for future experimental data.Therefore, for the standardization of W. ganpi, the chemical structures of three marker compounds (two flavonoids and one coumarin) isolated from the methanol (MeOH) extract were identified using NMR, and the HPLC-PDA analysis was developed and validated according to the ICH guidelines.
Herb-drug interactions (HDIs) are increasingly being recognized as important clinical factors that may alter the oral bioavailability of therapeutic agents co-administered with herbal medicines [29].Many of the major pharmacokinetic interactions between drugs are due to the effects of previous drug administration on hepatic cytochrome P450 (CYP) enzymes [30].In particular, the modulation of drug metabolic enzymes, such as CYP induction or inhibition, via herb-derived compounds is one of the well-recognized primary causes of HDIs [31,32].Several studies have shown that herbal medicines can inhibit CYP activity, causing serious HDIs when combined with conventional medicines [33].To the best of our knowledge, in vitro studies have evaluated the biological activity of W. ganpi MeOH extract (WGM), but no studies have evaluated the HDI potential of WGM itself.Therefore, the evaluation of HDIs associated with drug-metabolizing enzymes is necessary to ensure the safe use of herbal products.Herein, we report the direct effects of WGM and marker compounds on CYP-mediated drug metabolism in rats in vitro and in vivo.The inhibitory effect of the three marker compounds of WGM on CYP metabolic activity in the rat liver S9 fraction was evaluated for their half-maximal inhibitory concentrations (IC 50 ).The in vivo pharmacokinetic interaction was evaluated following a single oral administration of buspirone (BUS; probe substrate for CYP3A) [34,35] alone or co-administered with WGM in a rat model.

Plant Materials
W. ganpi were collected from Geumsa-ri, Yeongnam-myeon, Goheung-gun, Jeollanamdo, Republic of Korea, and the aerial parts were air-dried for use.They were identified by Jin-Hyub Paik (International Biological Material Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea).A voucher specimen marked as PNU-0027 has been deposited at the College of Pharmacy of Pusan National University (Busan, Republic of Korea).

Preparation of Sample and Reference Solutions
Powdered WGM was dissolved in DMSO/MeOH (50:50, v/v) at a concentration of 20.0 mg/mL and used as the sample stock solution.Reference compounds [7-methoxyluteolin-5-O-glucoside (1), pilloin 5-O-β-D-glucopyranoside (2), and rutarensin (3)] isolated from WGM were dissolved in DMSO/MeOH (50:50, v/v) at a concentration of 1200 µg/mL and used as reference stock solutions.Stock solutions were refrigerated at 4 • C, and working solutions were prepared and tested every time.

HPLC-PDA Method Validation
The developed HPLC-PDA simultaneous analysis conditions were validated according to ICH Q2(R1) guidelines [36].The validation characteristics included specificity, linearity, detection limit, quantitation limit, precision, recovery, and stability.Specificity is defined as the ability to clearly identify an analyte in a sample containing a mixture of impurities.This was verified by comparing and overlaying the chromatograms and PDA spectra obtained from the WGM samples and the reference mixture to confirm that the retention time (t R ), spectral pattern, and maximum absorption wavelength (λ max ) of the three reference compounds were consistent.Linearity is a test used to derive calibration curves by obtaining measurements that are directly proportional to the concentration of the substance being examined within a certain range.The stock solutions of 7-methoxyluteolin-5-O-glucoside (1), pilloin 5-O-β-D-glucopyranoside (2), and rutarensin (3) were diluted to six target concentrations (5.0, 50.0, 250.0, 400.0, 500.0, and 600.0 µg/mL) in DMSO/MeOH (50:50, v/v) mixtures, and six replicates (n = 6) were performed for each working solution.The calibration curve was expressed using a linear equation (y = ax + b) through linear regression analysis as a function of the area (y) vs. the concentration (x) of each reference peak, and the slope (a), y-intercept (b), and correlation coefficient (r 2 ) were calculated.The correlation coefficients for the acceptance criteria exceeded 0.999.The detection limit (DL) and quantitation limit (QL) are the minimum detection and quantitation limits of the analyte in the sample, respectively, that can be reliably quantified.These values were measured using the standard deviation (SD) of the y-intercept (σ) and the slope of the calibration curve (S) derived based on the linearity evaluation and were calculated as follows: DL = 3.3 σ/S and QL = 10 σ/S.Precision is an analytical procedure used to evaluate repeatability and reproducibility.The repeatability of the experiment was assessed by measuring the intraand inter-day precision.Three reference compounds were repeated five times (n = 5) at low, medium and high concentration levels (10.0, 100.0, and 300.0 µg/mL).Intra-day precision was injected on the same day, while inter-day precision was injected on days 1, 3, and 5.The results were determined using the relative standard deviation (RSD) of the peak area.The %RSD was calculated as follows: RSD (%) = (standard deviation/mean measured amount) × 100.%RSD value of <2% means that the developed HPLC-PDA method is precise and reproducible enough for simultaneous analysis and evaluation of the three compounds.A recovery test is conducted to verify the accuracy of the proposed method.%Recovery was determined by spiking each reference solution at three concentration levels (12.0, 60.0, and 120.0 µg/mL) into the WGM sample.The analysis was repeated three times (n = 3), and the measured values were calculated as follows: % recovery = (found concentration-original concentration)/spiked concentration × 100.Stability was used to evaluate the degree of change in the solution concentration with storage conditions and time.The stock solution of the WGM samples and the three reference compounds was maintained at room temperature (R.T., 22 ± 3 • C) and 4 • C for 72 h, and the measurements were taken five times (n = 5) at 6, 24, 48, and 72 h.The stability results were evaluated as the %peak area change and %RSD between the mean peak area of the control group (0 h) and that of the experimental group (6, 24, 48, and 72 h).

In Vitro Metabolic Inhibition Study in Rat Liver S9 Fraction
The composition of the reaction mixture used for in vitro CYP metabolic inhibition studies was as follows: 100 mM potassium phosphate buffer, 10 mM MgCl 2 , 1 mM NADPH, rat liver S9 fraction (1 mg/mL), CYP isoforms-specific probe substrate, and 50 µg/mL WGM.CYP isoforms-specific probe substrates were BUS for CYP3A, DIC for CYP2C, and DEX for CYP2D [34,35,37].Dose-response curves assessed metabolic inhibition of BUS in the presence of 1 µM BUS and 10 different concentrations (0, 0.1, 0.5, 1, 2, 5, 10, 20, 50, and 100 µM) of 7-methoxyluteolin-5-O-glucoside, pilloin 5-O-β-D-glucopyranoside or rutarensin.The mixture without substrate and inhibitor was incubated for 5 min at 37 • C, after which the substrate and inhibitor were added to initiate the enzyme reaction [38,39].After incubating BUS (reaction time: 30 min), DIC and DEX (reaction time: 60 min) at 37 • C at a speed of 500 oscillations/min, a sample was obtained from the reaction mixture and mixed with cold acetonitrile containing an internal standard (IS) to terminate the reaction.After sample preparation, the samples were analyzed using the ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method (see the sections entitled 'Biological Sample Preparation' and 'UPLC-MS/MS Conditions' in the Supplementary Materials).

In Vivo Pharmacokinetic Study in Rats
To evaluate the effects of WGM on the pharmacokinetics of BUS in vivo, 12 rats underwent surgical cannulation of the femoral artery after anesthetization via an intramuscular injection of 10 mg/kg zoletil [40,41].After sufficient recovery from anesthesia, 6 rats were administered a single oral dose of BUS (30 mg/kg) and 6 rats were administered WGM (1 g/kg) together with BUS.Approximately 150 µL blood samples were collected from a cannula inserted into the femoral artery at 0, 2, 5, 10, 15, 30, 60, 120, 180, 240, 360, and 480 min post-administration.The blood was immediately centrifuged at 3000× g for 10 min at 4 • C to obtain the plasma [42], which was pretreated and analyzed using the UPLC-MS/MS method.

Pharmacokinetic Analysis and Statistic
The half-maximal drug inhibitory concentration (IC 50 ) of the WGM marker compounds for the metabolism of BUS was determined using the four-parameter logistic equation indicated as follow (GraphPad Prism ver.5.01; San Diego, CA, USA): where x is inhibitor concentration, y is response, Max is the initial y values, Min is the final y values, and P is the Hill coefficient.The average plasma pharmacokinetic parameters, including the area under the curve from time 0 extrapolated to infinity (AUC inf ) and the terminal half-life (t 1/2 ), were determined using a non-compartment model (WinNonlin Ver.3.1; Certara, Inc., Princeton, NJ, USA) [43].The peak time (T max ) and maximal concentration (C max ) were obtained directly from the observed plasma concentration-time profiles.A t-test was used to compare two unpaired means, and a p-value of below 0.05 was considered statistically significant.

Development of HPLC-PDA Conditions
HPLC-PDA was used to develop a method for simultaneous analysis of the three marker components isolated from WGM.The developed method should be able to rapidly analyze the target components with high resolution.Therefore, the analysis conditions were optimized by considering column temperature, mobile phase, gradient conditions,

Development of HPLC-PDA Conditions
HPLC-PDA was used to develop a method for simultaneous analysis of the three marker components isolated from WGM.The developed method should be able to rapidly analyze the target components with high resolution.Therefore, the analysis conditions were optimized by considering column temperature, mobile phase, gradient conditions, flow rate, and wavelength.For the mobile phase, 0.1% FA was added to acetonitrile (A) and water (B) to increase resolution.The gradients were first tested with the WGM sample solution under the conditions of 0-60 min and 5-95% A. When separation was performed under the first condition, the three marker components [7-methoxyluteolin-5-O-glucoside (1), pilloin 5-O-β-D-glucopyranoside (2), and rutarensin (3)] were detected.However, the retention time of 7-methoxyluteolin-5-O-glucoside (1) was relatively long (approximately 20 min), and pilloin 5-O-β-D-glucopyranoside (2) and rutarensin (3) could not be properly separated.To improve resolution and shorten analysis time, a multi-step gradient was applied, and the temperature (e.g., 30, 35, and 40 • C) and flow rate (e.g., 0.7 and 1.0 mL/min) were set.The analysis wavelength was selected by scanning the 210-400 nm range using PDA.Finally, a gradient elution system of 0.0-5.0min, 13-20% A; 5-23 min, 20-30% A; 23-25 min, 30-70% A; 25-30 min, 70-70% A, was used.The optimized conditions successfully separated the three marker components of the WGM sample, which were detected within 30 min, as shown in Figure 2.

Development of HPLC-PDA Conditions
HPLC-PDA was used to develop a method for simultaneous analysis of the three marker components isolated from WGM.The developed method should be able to rapidly analyze the target components with high resolution.Therefore, the analysis conditions were optimized by considering column temperature, mobile phase, gradient conditions, flow rate, and wavelength.For the mobile phase, 0.1% FA was added to acetonitrile (A) and water (B) to increase resolution.The gradients were first tested with the WGM sample solution under the conditions of 0-60 min and 5-95% A. When separation was performed under the first condition, the three marker components [7-methoxyluteolin-5-O-glucoside (1), pilloin 5-O-β-D-glucopyranoside (2), and rutarensin (3)] were detected.However, the retention time of 7-methoxyluteolin-5-O-glucoside (1) was relatively long (approximately 20 min), and pilloin 5-O-β-D-glucopyranoside (2) and rutarensin (3) could not be properly separated.To improve resolution and shorten analysis time, a multi-step gradient was applied, and the temperature (e.g., 30, 35, and 40 °C) and flow rate (e.g., 0.7 and 1.0 mL/min) were set.The analysis wavelength was selected by scanning the 210-400 nm range using PDA.Finally, a gradient elution system of 0.0-5.0min, 13-20% A; 5-23 min, 20-30% A; 23-25 min, 30-70% A; 25-30 min, 70-70% A, was used.The optimized conditions successfully separated the three marker components of the WGM sample, which were detected within 30 min, as shown in Figure 2.

Validation of Methods
Specificity was evaluated by comparing the HPLC-PDA chromatograms obtained from the WGM samples and the reference mixture.In the WGM samples and reference mixture, the retention time (tR) and maximum absorption wavelength (λmax) of the three compounds were as follows: 7-Methoxyluteolin-5-O-glucoside (tR of 13.886 min, and λmax at 241.3 and 341.4 nm), pilloin 5-O-β-D-glucopyranoside (tR of 19.280 min, and λmax at 242.5 and 340.2 nm), and rutarensin (tR of 24.088 min, and λmax at 336.6 nm).In addition, as shown in Figure 3, the PDA spectra matched with each other.As a result, all parameters [retention time (tR), PDA spectra, and maximum absorption wavelength (λmax)] of the WGM samples and reference mixture were matched.Therefore, the specificity of the three

Validation of Methods
Specificity was evaluated by comparing the HPLC-PDA chromatograms obtained from the WGM samples and the reference mixture.In the WGM samples and reference mixture, the retention time (t R ) and maximum absorption wavelength (λ max ) of the three compounds were as follows: 7-Methoxyluteolin-5-O-glucoside (t R of 13.886 min, and λ max at 241. 242.5 and 340.2 nm), and rutarensin (t R of 24.088 min, and λ max at 336.6 nm).In addition, as shown in Figure 3, the PDA spectra matched with each other.As a result, all parameters [retention time (t R ), PDA spectra, and maximum absorption wavelength (λ max )] of the WGM samples and reference mixture were matched.Therefore, the specificity of the three compounds was validated, and the developed method allowed for the selective and accurate analysis of the analyte.

Validation of Methods
Specificity was evaluated by comparing the HPLC-PDA chromatograms obtained from the WGM samples and the reference mixture.In the WGM samples and reference mixture, the retention time (tR) and maximum absorption wavelength (λmax) of the three compounds were as follows: 7-Methoxyluteolin-5-O-glucoside (tR of 13.886 min, and λmax at 241.3 and 341.4 nm), pilloin 5-O-β-D-glucopyranoside (tR of 19.280 min, and λmax at 242.5 and 340.2 nm), and rutarensin (tR of 24.088 min, and λmax at 336.6 nm).In addition, as shown in Figure 3, the PDA spectra matched with each other.As a result, all parameters [retention time (tR), PDA spectra, and maximum absorption wavelength (λmax)] of the WGM samples and reference mixture were matched.Therefore, the specificity of the three compounds was validated, and the developed method allowed for the selective and accurate analysis of the analyte.Linearity was verified by analyzing the three marker components in the concentration range of 5.00 to 600.00 µg/mL.Each reference compound was tested six times at each concentration to obtain calibration data at six concentrations.The regression equation and Linearity was verified by analyzing the three marker components in the concentration range of 5.00 to 600.00 µg/mL.Each reference compound was tested six times at each concentration to obtain calibration data at six concentrations.The regression equation and coefficient of determination (r 2 ) for the calibration curve are listed in Table 1.The r 2 values of all the curves showed good linearity, with r 2 > 0.999 across the concentration range used.
Table 1.Linear range, regression equation, coefficient of determination, detection limit, and quantitation limit of three marker components (n = 6).

Compound a
Linear Range (µg/mL)  The DL and QL were between 0.72-3.34µg/mL and 2.19-10.12µg/mL, respectively.The results for each component are shown in Table 1.
The intra-and inter-day %RSD for the three marker components at low, medium, and high concentrations ranged from 0.26-1.55%and 0.60-1.68%,respectively.Among the three marker components, the %RSD values for the intra-and inter-day of pilloin 5-O-β-D-glucopyranoside (2) were slightly higher.However, as shown in Table 2, the total repeatability of all compounds did not exceed 2%; thus, the precision was validated according to the ICH Q2(R1) guidelines.To verify the accuracy of the method by evaluating the closeness of the measured values to the actual values, known concentrations of the three marker components were spiked into the WGM samples, and the %recovery was calculated using repeated (n = 3) measurements of the analyte.As shown in Table 3, the average %recovery for 12.0, 60.0, and 120.0 µg/mL of the three marker components was within 93.42-117.55%,and the %RSD values were below 2%.In other words, the %recovery was the lowest when the medium concentration of rutarensin (3) was spiked and the highest when the medium concentration of 7-methoxyluteolin-5-O-glucoside (1) was spiked.The stability of the three marker components to storage conditions (R.T. and 4 • C) was evaluated by calculating the %difference and %RSD.Under all storage conditions, the three compound peak areas of the WGM sample solution increased after 6 h and decreased after 48 h.After 72 h, the peak areas of the three compounds increased by 0.99-3.56%from 0 h under all storage conditions.In addition, the %RSD was less than 2%, and stability was confirmed under all conditions.In the reference solution, the peak areas of the three compounds decreased after 6 h in 4 • C storage conditions.Under room temperature storage conditions, 7-methoxyluteolin-5-O-glucoside (1) and rutarensin (3) decreased by 0.54 and 3.23%, respectively, but pillion 5-O-β-D-glucopyranoside (2) increased by 1.71%.After 48 h, pillion 5-O-β-D-glucopyranoside (2) increased by 7.58 and 4.42% under conditions R.T. and 4 • C, respectively, showing an unstable state.Consequently, it is recommended to use the WGM sample and reference solution within 6 h after preparation and to store it at 4 • C. The stabilities of the three compounds in the WGM sample and reference solutions are detailed in Table 4.

Chemical Profiling of WGM
Quantitative analysis of the three marker components in the WGM samples was performed using the developed and validated HPLC-PDA assay.The assay was repeated five times (n = 5), and the content of each compound was calculated using a calibration curve.The three marker components in the WGM sample were measured to be within 17.10-51.81mg/g.The most abundant marker compound in W. ganpi was 7-methoxyluteolin-5-O-glucoside (1) (Table 5).The inhibitory effects of WGM on CYP activity were assessed by measuring the disappearance of various CYP substrates in the rat liver S9 fraction, as shown in Figure 4a.The metabolism of DEX and DIC did not change in the presence of 50 µg/mL WGM; however, it significantly inhibited the metabolism of BUS in the presence of 50 µg/mL WGM compared to the control group (p = 0.000169 for BUS).The inhibitory effect of CYP at various concentrations (0-100 µM) of WGM marker compound on metabolism of BUS in rat liver S9 fraction was evaluated through the construction of a dose-response curve (Figure 4b-d).7-Methoxyluteolin-5-O-glucoside and pilloin 5-O-β-D-glucopyranoside, among the marker compounds of WGM, inhibited BUS metabolism in the rat liver S9 fraction with IC 50 values of 2.73 and 18.7 µM, respectively.However, rutarensin did not inhibit BUS metabolism.These results indicate that WGM inhibits the metabolic activity of CYP3A but not CYP2C and CYP2D [37,46,47] and that the inhibition of CYP3A activity via WGM is attributed to 7-methoxyluteolin-5-O-glucoside and pilloin 5-O-β-D-glucopyranoside. liver S9 fraction was evaluated through the construction of a dose-response curve (Figure 4b-d).7-Methoxyluteolin-5-O-glucoside and pilloin 5-O-β-D-glucopyranoside, among the marker compounds of WGM, inhibited BUS metabolism in the rat liver S9 fraction with IC50 values of 2.73 and 18.7 µM, respectively.However, rutarensin did not inhibit BUS metabolism.These results indicate that WGM inhibits the metabolic activity of CYP3A but not CYP2C and CYP2D [37,46,47] and that the inhibition of CYP3A activity via WGM is attributed to 7-methoxyluteolin-5-O-glucoside and pilloin 5-O-β-D-glucopyranoside.

In Vivo Pharmacokinetic Studies in Rats
The mean plasma concentration versus time profiles following the oral administration (30 mg/kg) of BUS with or without concomitant oral administration of WGM (1 g/kg) in rats are shown in Figure 5.The corresponding pharmacokinetic parameters of BUS with or without WGM administration are presented in Table 6.The Tmax of BUS was 5-15 min, showing rapid absorption into the systemic circulation.Compared to the control group, the WGM group did not show significant changes in the pharmacokinetic parameters of the administered BUS (p > 0.05).The inhibitory effects of WGM on various CYP metabolic activities were assessed by comparing the disappearance of the substrate (BUS, DEX, and DIC) between the absence and presence of 50 µg/mL WGM.Although the metabolic activity of BUS was significantly decreased in the rat liver S9 fraction, the activities of DEX and DIC did not significantly decrease in the presence of WGM.Among the marker components of WGM, 7-methoxyluteolin-5-O-glucoside and pilloin 5-O-β-D-glucopyranoside exhibited inhibitory effects on BUS metabolism in the rat liver S9 fraction with IC50 values of 2.73 and 18.7 µM, respectively.However, rutarensin did not inhibit BUS metabolic activity.This suggests that 7-methoxyluteolin-5-O-glucoside and pilloin 5-O-β-D-glucopyranoside regulate the metabolism of CYP3A [47,48].Based on the results of this experiment, the pharmacokinetics were investigated after the oral administration of BUS with or without WGM in rats.The group administered simultaneously with WGM increased AUCinf and Cmax by 21.0% and 23.7% compared to the control group, but there was no significant difference between the two groups.BUS is primarily eliminated via hepatic metabolism in rats and humans [35].In vitro CYP inhibition studies using rat liver S9

In Vivo Pharmacokinetic Studies in Rats
The mean plasma concentration versus time profiles following the oral administration (30 mg/kg) of BUS with or without concomitant oral administration of WGM (1 g/kg) in rats are shown in Figure 5.The corresponding pharmacokinetic parameters of BUS with or without WGM administration are presented in Table 6.The T max of BUS was 5-15 min, showing rapid absorption into the systemic circulation.Compared to the control group, the WGM group did not show significant changes in the pharmacokinetic parameters of the administered BUS (p > 0.05).The inhibitory effects of WGM on various CYP metabolic activities were assessed by comparing the disappearance of the substrate (BUS, DEX, and DIC) between the absence and presence of 50 µg/mL WGM.Although the metabolic activity of BUS was significantly decreased in the rat liver S9 fraction, the activities of DEX and DIC did not significantly decrease in the presence of WGM.Among the marker components of WGM, 7-methoxyluteolin-5-O-glucoside and pilloin 5-O-β-D-glucopyranoside exhibited inhibitory effects on BUS metabolism in the rat liver S9 fraction with IC 50 values of 2.73 and 18.7 µM, respectively.However, rutarensin did not inhibit BUS metabolic activity.This suggests that 7-methoxyluteolin-5-O-glucoside and pilloin 5-O-β-D-glucopyranoside regulate the metabolism of CYP3A [47,48].Based on the results of this experiment, the pharmacokinetics were investigated after the oral administration of BUS with or without WGM in rats.The group administered simultaneously with WGM increased AUC inf and C max by 21.0% and 23.7% compared to the control group, but there was no significant difference between the two groups.BUS is primarily eliminated via hepatic metabolism in rats and humans [35].In vitro CYP inhibition studies using rat liver S9 suggest that BUS, a substrate of CYP3A, reduced liver-specific clearance (CL int,H ).However, there were no significant changes in the pharmacokinetic parameters of BUS in vivo with or without WGM, suggesting that there was no change in the liver-specific clearance of BUS.Several factors might have contributed to these findings.One of the causes may be low bioavailability due to chemical degradation of phytochemicals and the intestinal/hepatic first pass [38,41,49,50], and plasma and liver concentrations of inhibitors may be insufficient for the IC 50 obtained in vitro CYP inhibition studies [51,52].Consequently, a single oral dose of WGM did not have significant effects on the pharmacokinetics of buspirone in rats, suggesting that WGM cannot function as an inhibitor of CYP3A-mediated metabolism in vivo.This should consider the interspecies differences between rats and humans.Further studies are required to confirm whether WGM is a clinically relevant CYP3A4 inhibitor.
Nutrients 2023, 15, x FOR PEER REVIEW 13 of 16 and humans.Further studies are required to confirm whether WGM is a clinically relevant CYP3A4 inhibitor.

16 Figure 1 .
Figure 1.Chemical structures of the three compounds isolated from WGM.

Figure 1 .
Figure 1.Chemical structures of the three compounds isolated from WGM.

Figure 1 .
Figure 1.Chemical structures of the three compounds isolated from WGM.

Figure 4 .
Figure 4. Effects of WGM on the disappearance rate of model cytochrome P450 substrate in rat liver S9 fraction (a).Dose-response curves for the inhibitory effect of 7-methoxylutolin-5-O-glucoside (b) pilloin 5-O-β-D-glucopyranoside (c), and rutarensin (d) on the disappearance of BUS in rat liver S9 fraction (n = 5).The asterisk represents a value significantly different from that of the other groups (p < 0.05).

Figure 5 .
Figure 5.The mean plasma concentration-time curve of BUS after oral administration of 30 mg/kg in rats (green circles) or following co-administration with 1 g/kg WGM (red circles).Symbols show means and error bars represent SD (n = 6).

Table 2 .
Intra-and inter-day variabilities of three marker components.

Table 4 .
Stabilities of three marker components in WGM and reference solutions (n = 5).

Table 6 .
Pharmacokinetic parameters of BUS after oral administration of 30 mg/kg in rats or following co-administration with 1 g/kg WGM.Data are expressed as means ± SD (n = 6).