Hepatic, gastric, and intestinal first-pass effects of vitexin in rats

Abstract

Context: Recent research has demonstrated that vitexin exhibits a prominent first-pass effect. In this light, it is necessary to investigate the causes of this distinct first-pass effect.

Objective: The aim of this study was to evaluate hepatic, gastric, and intestinal first-pass effects of vitexin in rats and, furthermore, to investigate the role of P-glycoprotein (P-gp) and cytochrome P450 3A (CYP3A) in the absorption and secretion of vitexin in the duodenum.

Materials and methods: Vitexin was infused into rats intravenously, intraportally, intraduodenally, and intragastrically (30 mg/kg). In addition, verapamil (50 mg/kg), a common substrate/inhibitor of P-gp and CYP3A, was also instilled with vitexin into the duodenum to investigate the regulatory action of P-gp and CYP3A. The plasma concentrations of vitexin were measured by the HPLC method using hesperidin as an internal standard.

Results: The hepatic, gastric, and intestinal first-pass effects of vitexin in rats were 5.2%, 31.3%, and 94.1%, respectively. In addition, the total area under the plasma concentration–time curve from zero to infinity (AUC) of the vitexin plus verapamil group and of the normal saline group was 44.9 and 39.8 μgċ min/mL, respectively.

Discussion and conclusion: The intestinal first-pass effect of vitexin was considerable, and gastric and hepatic first-pass effects also contribute to the low absolute oral bioavailability of vitexin. The AUC of the vitexin plus verapamil group was slightly higher than that of the vitexin plus normal saline group (by approximately 1.13-fold), suggesting that verapamil does not play an important role in the absorption and secretion of vitexin.

• HPLC
• pharmacokinetics
• verapamil

Introduction

Flavonoids are ubiquitously distributed in the plant kingdom, and they present numerous biological and pharmacological activities, as they are effective in the treatment of inflammation, heart disease, and cancer (Benavente-Garcia & Castillo, 2008; Hollman et al., 1985; Middleton et al., 2000). Vitexin, a main compound in hawthorn fruits and leaves, belongs to a class of flavonoids, and it has been shown to possess many pharmacological activities including hypotensive, anti-inflammatory, and antispasmodic (non-specific) properties; it also exhibits antithyroid effects, antimicrobial effects, and demonstrates significant inhibitory activities in anti-glycation due to its free radical scavenging activity (Gaitan et al., 1995; Peng et al., 2008; Prabhakar et al., 2007). Therefore, many scholars have paid more attention to vitexin, especially based on its pharmacokinetic properties in beagles, mice, and rats after single- and multi-dose intravenous administration of its monomer and extract (Tong & Liu, 2006, 2007; Wang et al., 2010). In addition, the tissue distribution and excretion of vitexin have also been investigated (Wang et al., 2012b), and it has been found that vitexin exhibited very low absolute oral bioavalibility (F) in rats (Wang et al., 2012a), as it exhibited a prominent first-pass effect; however, little research has been done on the first-pass effect of vitexin. The hepatic, gastric, and intestinal first-pass effects, which are the three main processes responsible for these effects, were therefore investigated using the rat’s first-pass effect model in the study. In addition, verapamil, a common substrate/inhibitor of P-glycoprotein (P-gp) and cytochrome P450 3A (CYP3A), was selected to evaluate the effect it has on the intestinal absorption of vitexin, as it would clarify whether the absorption and secretion of vitexin are mediated by P-gp and CYP3A (Saitoh & Aungst, 1995; Thummel et al., 1997; Wandel et al., 1999).

Materials and methods

Materials

Vitexin [>99% by high-performance liquid chromatography (HPLC) analysis] was used for oral and intravenous administration; it was isolated from the leaves of Crataegus pinnatifida Bge. var. major N. E. Brown (Rosaceae) in our laboratory. The leaves, identified by Professor Tingguo Kang, were collected on 18 September 2011 in Shenyang, Liaoning Province, China, and the voucher specimen (Number 20110918) is maintained at the Liaoning University of Traditional Chinese Medicine. The chemical structure of vitexin, confirmed by ¹H- and ¹³C-nuclear magnetic resonance spectroscopy, is shown in Figure 1(a).

Figure 1. Chemical structures of vitexin (a) and hesperidin (b).

Hesperidin [Batch number 110753-200413, the chemical structure is shown in Figure 1(b)], acting as an internal standard (I.S.), was provided by the National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China. Verapamil hydrochloride was obtained from the Central Pharmaceutical Co. Ltd. (Tianjin, China). The water used in all the experiments was purified by a Milli-Q® Biocel Ultrapure Water System (Millipore, Bedford, MA). Methanol and acetonitrile were of HPLC grade, and all other reagents were of analytical grade.

Animals

Male Wistar rats weighing 280–330 g were obtained from the Laboratory Animal Center of Liaoning University of Traditional Chinese Medicine (Shenyang, China). All rats were kept in a controlled environment and had free access to standard laboratory food and water intake for a week. The rats were fasted for 12 h prior to administration, but they had free access to water. All animal studies were performed according to the Guidelines for the Care and Use of Laboratory Animals, which was approved by the Committee of Ethics of Animal Experimentation of Liaoning University of Traditional Chinese Medicine.

Hepatic first-pass effect

Rats were anesthetized with 20% urethane (0.3 mL/kg i.p.) to accept pre-disposal, and their body temperature was maintained by infrared light irradiation during the entire experiment. The carotid artery was catheterized with polyethylene tubing filled with heparinized saline (80 U/mL) for the collection of blood.

For intravenous administration (n = 5), the femoral vein was exposed in order to cannulate polyethylene tubing to infuse a 1.5 mL vitexin solution (30 mg/kg) within 5 min. For intraportal administration (n = 5) (Braillon & Brody, 1988), the abdominal cavity was opened with a midline incision to find the superior mesenteric vein along the bowel; the cannula was advanced gently into the superior mesenteric vein, and a 1.5 mL vitexin solution (30 mg/kg) was also infused into the portal vein within 5 min.

Blood samples were collected into heparinized tubes via the carotid artery at time 0 (before infusion), 2, and 5 min (the terminal point of the infusion), and again at 8, 11, 15, 20, 30, 45, 60, 90, 120, and 180  min after the administration of vitexin. The samples were then centrifuged at 890 g for 15 min. The obtained plasma samples were stored at −20 °C until analysis.

Gastric and intestinal first-pass effects

For intraportal infusion (n = 5), 1.5 mL of vitexin solution (30 mg/kg) was infused into the portal vein within 5 min according to the method used to assess the hepatic first-pass effect, and a 1.5 mL aliquot of normal saline was instilled into the stomach and duodenum. For intraduodenal administration (n = 5), the upper site of the duodenum (1 cm below the pylorus on the small intestine) was exposed to infuse 1.5 mL of vitexin solution (30 mg/kg) within 5 min, and 1.5 mL aliquots of normal saline were instilled into the stomach and the portal vein within 5 min. For intragastric administration (n = 5), 1.5 mL of vitexin solution (30 mg/kg) was instilled into the stomach at the bottom of the cardia within 5 min; at the same time, 1.5 mL aliquots of normal saline were also instilled into the portal vein and the duodenum. Blood samples were collected via the carotid artery, and the samples were processed and stored in a method similar to that of the hepatic first-pass effect.

Verapamil hydrochloride on intestinal absorption of vitexin

To investigate the effect of P-gp and CYP3A on the intestine, 2 mL of verapamil hydrochloride (50  mg/kg) solution was instilled into duodenum 10 min in advance before the perfusion of the vitexin solution (30 mg/kg; n = 5) among the group receiving vitexin with verapamil. At the same time, an equal volume of normal saline was infused into the vitexin group in the similar manner. The other procedures that were conducted were the same as those methods mentioned above for the hepatic first-pass effect.

Sample processing

To the 100 μL of plasma sample, 10 μL of acetic acid, 20 μL of I.S. (hesperidin, 162 μg/mL), and 500 μL of acetonitrile were successively pipetted, followed by vortex mixing for 1 min and centrifugation at 890 g for 15 min. The supernatant was separated and evaporated to dryness under a stream of nitrogen at 40 °C. The residue was diluted in 100 μL of mobile phase and centrifuged at 15092  g for 5 min. A 20  μL aliquot of each supernatant was analyzed by HPLC.

Chromatography system

The analyses were carried out on an Agilent 1100 series HPLC system (Agilent Technologies, Palo Alto, CA), which consisted of a quaternary pump (G1310A), a vacuum degasser (G1322A), an ultraviolet (UV)–visible spectrophotometric detector (G1314A), and Chemstation software (Agilent Technologies, Palo Alto, CA). The analytes were determined at 25 °C, using a column heater (Replete Technology, Dalian, China) kept on an analytical Diamonsil C₁₈ column (150 mm × 4.6 mm i.d., 5 μm, Dalian Create Science and Technology Co., Ltd., Beijing, China), protected by a KR C₁₈ guard column (35 mm × 8.0 mm, i.d., 5 μm, Dalian Create Science and Technology Co., Ltd., Beijing, China). The mobile phase, which consisted of methanol–acetonitrile–0.3% aqueous formic acid (3:1:6, v/v/v), was filtered and degassed under reduced pressure before use. All chromatographic measurements were performed at 25 °C, at a flow rate of 1 mL/min, and with the detection wavelength of 330 nm.

Preparation of standards and quality control samples

Standard stock solutions of vitexin and I.S. were prepared in methanol to yield the concentrations of 500 and 324 μg/mL, respectively. The working solutions were prepared with stepwise diluted stock solution to concentrations over the range of 1–500 μg/mL. All the working solutions were stocked at 4 °C. Eight calibrators (0.1–50 μg/mL) of vitexin were prepared by adding standard working solutions (10 μL) and working solution I.S. (162 μg/mL, 20 μL) to drug-free rat plasma.

The quality control (QC) samples were prepared at low (0.3 μg/mL, three times the lower limit of quantitation), high (40 μg/mL, 80% of the upper limit of quantitation), and middle (3.5 μg/mL, near the geometric mean of low and high concentrations) concentrations in bulk, and aliquots were stored at −20 °C prior to analysis.

For method validation, the accuracy, precision, extraction efficiency, short-term stability, long-term stability, and freeze-thaw stability were evaluated with QC samples at three concentrations (CDER & CVM, 2001). The QC samples of vitexin were prepared in a similar manner as the standard samples.

Pharmacokinetic study and statistical analysis

Pharmacokinetic data were processed by non-compartmental methods with the assistant 3p97 software (The Chinese Society of Mathematical Pharmacology, Beijing, China). Then, the data were statistically analyzed by SPSS software (IBM Corporation, Armonk, NY), and the differences were considered significant when p < 0.05. All data were expressed as the mean ± standard deviation.

Results

HPLC assay

The evaluation of linearity was performed with an eight-point calibration curve over the concentration range of 0.1–50 μg/mL. The slope and intercept of the calibration graphs were calculated by weighted (1/c²) least squares linear regression. The regression equation of the calibration curves was typically: y = 0.2384x + 0.0019, where r²= 0.9967, y is the peak area ratio of vitexin to I.S., and x is the plasma concentration of vitexin. Method validation, including accuracy, precision, extraction efficiency, short-term stability, long-term stability, and freeze-thaw stability evaluated with QC samples at three concentrations were within the acceptable range (CDER & CVM, 2001).

Measurement of hepatic first-pass effect

The plasma concentration–time curve of vitexin after intravenous and intraportal administration is shown in Figure 2(a), and the pharmacokinetic parameters are listed in Table 1. The hepatic first-pass effect of vitexin after absorption into the portal vein was 5.2% in rats, and it was calculated using the following formula:

Figure 2. Mean plasma concentration–time curves of vitexin after intravenous (○; n = 5) and intraportal (•; n = 5) administration (a), and intragastric (▪; n = 5), intraduodenal (□; n = 5), and intraportal (•; n = 5) administration (b) (mean ± SD).

Table 1. Pharmacokinetic parameters of vitexin after intravenous, intraportal, intragastric and intraduodenal administration with or without administration of verapamil (n = 5).

	Hepatic first-pass effect		Gastric and intestinal first-pass effects			Intestinal first-pass effect
Parameter	Intravenous	Intraportal	Intraportal	Intragastric	Intraduodenal	Intraduodenal (vitexin + NS)	Intraduodenal (vitexin + verapamil)
AUC (μg·min/mL)	774.3 ± 23.7^a	734.2 ± 18.4	719.4 ± 23.5	29.2 ± 1.4^b	42.5 ± 4.9	39.8 ± 2.2	44.9 ± 1. 6^c
First-pass effect (%)	–	5.2	–	31.3	94.1	–	–

^aIn hepatic first-pass effect, the intravenous group was significantly different (p < 0.01) from the intraportal group.

^bIn gastric and intestinal first-pass effects, the intragastric group was significantly different (p < 0.001) from the intraduodenal group.

^cIn intestinal first-pass effect, vitexin with verapamil group was significantly different (p < 0.01) from vitexin with NS group.

Measurement of gastric and intestinal first-pass effects

The plasma concentration–time curves of vitexin following intraportal, intragastric, and intraduodenal administration are shown in Figure 2(b). The pharmacokinetic parameters are listed in Table 1, in which the area under the curve (AUC) values of vitexin following intragastric and intraduodenal administration were significantly smaller than that after intraportal administration. The first-pass effects obtained for the intestine and stomach were 94.1 and 31.3%, respectively, and they were calculated using the following formulae:

Effect of verapamil on the absorption of vitexin in the intestine

The plasma concentration–time curve of vitexin following intraduodenal administration with pre-instillation of the verapamil hydrochloride and normal saline groups is shown in Figure 3. The pharmacokinetic parameters are listed in Table 1, and the AUC value of vitexin in the verapamil group was only a little higher than that of the normal saline group (by approximately 1.13-fold).

Figure 3. Mean plasma concentration–time curve of vitexin after intraduodenal administration with pre-instillation of verapamil (▴; n = 5) and normal saline (△; n = 5) (mean ± SD).

Discussion

To obtain a suitable retention time and good separation for the analysis, the mobile phase, consisting of methanol:acetonitrile:0.3% aqueous formic acid (3:1:6, v/v/v), was chosen after numerous trials. The UV absorption spectra of vitexin have two maximum absorptions at 269 and 331 nm, and that of I.S. at 204 and 284 nm. Interference from endogenous substances in the plasma was observed when the wavelength was set at 270 nm, and vitexin and the I.S. were readily separated from other endogenous peaks when the detection wavelength was set at 330 nm. To simultaneously acquire high extraction recovery and to enhance the precision of vitexin and I.S., several solvents of different volumes (such as acetonitrile and methanol) were selected to precipitate the protein. The highest recovery finally occurred after 500 μL of acetonitrile was used. Then, 10, 20, and 30 μL of acetic acid were individually tried and added in the plasma to avoid the dissociation of analyte; a good peak shape was obtained after 10 μL of acetic acid was added in the plasma.

The hepatic, intestinal, and gastric first-pass effects were obtained by using the formulas mentioned above [Equations (1)–(3)]. The hepatic first-pass effect of vitexin was 5.2% when calculated with using Equation (1), that is, calculating the AUC between intravenous (774 μg·min/mL) and intraportal administration (734 μg·min/mL), suggesting that the hepatic first-pass effect of vitexin after absorption into the portal vein of rats was the lowest among the three first-pass effects. With respect to the intraduodenal administration, which consisted of the hepatic and intestinal first-pass effects, Equation (2) can be used to calculate these effects. In addition, for the gastric administration, which consisted of the hepatic, intestinal, and gastric first-pass effects, the gastric first-pass effect formula (3) was used for calculations. The AUC after intragastrical and intraduodenal infusions were 29.2 and 42.5 μg·min/mL, meaning that the gastric first-pass effect of vitexin should be considered because the values of the gastric and intestinal effects were significantly different based on the SPSS analysis; thus, the gastric and intestinal first-pass effect was calculated, respectively, and to be 31.3 and 94.1%.

Vitexin, a kind of C-glucoside, was rapidly removed from the blood, and its absolute oral bioavailability was very low, which could be induced by many factors such as the absorption and secretion of vitexin mediated by P-gp and CYP3A, and the poor solubility associated with impaired drug bioavailability (Zu et al., 2012). Most drugs were absorbed in the intestine, where a variety of transport proteins exist. P-gp, the transporter, can reduce the transmembrane transport of drugs through the mechanism of efflux pump, subsequently acting as the absorption barrier of certain drugs (Borst et al., 1999; Lee et al., 2001). Verapamil, the inhibitor of P-gp and CYP3A, was therefore selected to evaluate the effect of vitexin on intestinal absorption in this study. The AUC increased slightly from 39.8 to 44.9 μg·min/mL after pre-infusion of verapamil, suggesting that verapamil did not remarkably improve the poor absorption of vitexin in the intestine.

In conclusion, the intestinal first-pass effect of vitexin was considerable (approximately 94%), and the gastric and hepatic first-pass effects were 30% and 5%, respectively; first-pass effects in both these systems contribute to the low F of vitexin. In addition, verapamil (the inhibitor of P-gp and CYP3A) does not appear to play an important role in the intestinal first-pass effect of vitexin.

Declaration of interest

The authors report no declarations of interest. The study was supported by the Shenyang Science and Technology Planning Foundation (F13-194-9-00), China.