Abstract [Objectives] This study was conducted to investigate the pharmacokinetic characteristics of arctigenin from water extract of Fructus Arctii in piglets， understand its absorption， distribution， transformation and excretion in piglets， and provide theoretical reference for the development and clinical use of new veterinary drugs.
　　[Methods] Six healthy piglets， weighing （30.0 ± 5.0） kg， were selected and administered by gavage with a Fructus Arctii water extract at 1.0 g/kg·bw. Blood was collected from the anterior vena cava at different time points. The concentration of arctigenin in pig plasma was determined by HPLC. The main pharmacokinetic parameters were： absorption half-life （t1/2ka） （0.098±0.006） h， distribution half-life （t1/2α） （0.208±0.009） h， elimination half-life （t1/2β） （23.816±2.151） h， apparent volume of distribution （Vd） （32.212±4.033） L/kg， apparent volume of distribution （Vd） （32.212±4.033） L/kg， clearance （CLb） （2.384±1.589） L/（h·kg）， peak time （tmax） （0.251±0.011） h， peak concentration （Cmax） （0.560 ± 0.063） μg/ml， and the area under the curve （AUC） at the time of drug administration （9.620 ± 0.752） μg·h/ml.
　　[Results] After oral administration of the water extract powder of Fructus Arctii， arctigenin showed a two-compartment model with absorption in piglets， with the characteristics of fast absorption， wide distribution and slow elimination. Arctigenin might have a hepatoenteral circulation in piglets， and the drug effect could last for a long time.
　　[Conclusions] This study provides a theoretical basis and reference for the development and clinical use of new veterinary drugs.

Key words Fructus Arctii; Arctigenin; Piglets; Pharmacokinetics

Received： July 12， 2020  Accepted： September 23， 2020

Supported by Hubei Provincial Technical Innovation Major Project （2019AEE006）; Wuhan Animal and Poultry Chinese Herbal Medicine Development Engineering Technology Research Center （2014021511020460）; Hubei Provincial Central Government Guiding Local Science and Technology Development Fundation of China （2020ZYYD029）.

Bin HE （1988-）， male， P. R. China， senior engineer， devoted to research about animal husbandry and veterinary medicine.

*Corresponding author. 　　 Fructus Arctii is a commonly used traditional Chinese medicine， which is cold in nature， pungent and bitter in taste. Its meridian tropisms are lung and stomach. It has the effects of dispelling wind and heat， opening the inhibited lung-energy， and promoting eruption， and is clinically used to treat exogenous wind-heat， cough and asthma， sore throat and other symptoms[1]. The main active ingredients of Fructus Arctii are lignan compounds， including arctiin and arctigenin[2]， of which arctiin is transformed into arctigenin under the action of gastrointestinal microorganisms to exert pharmacological effects[3]. That is to say， arctigenin is the direct active ingredient of Fructus Arctii[4-5]. Arctigenin has anti-inflammatory[6]， antiviral[7-8]， antibacterial[9]， antitumor[10-11]， liver protection[13]， antidiabetic[14] and many other pharmacological effects. It has good research and development and application prospects. In this study， the pharmacokinetic characteristics of Fructus Arctii water extract in piglets after intragastric administration were investigated， providing a theoretical basis and reference for the development and clinical use of new veterinary drugs and the exploration of the modernization of Chinese veterinary drugs.

Materials and Methods

Experimental materials

Agents
　　Arctigenin standard product （batch number： 160509） content≥98.1%， produced by Shanghai Winherb Medical Technology Co.， Ltd.， was accurately weighed， dissolved in methanol and diluted with water to constant volume， obtaining a standard stock solution containing arctigenin 1.0 mg/ml. The arctigenin standard was accurately weighed， dissolved in propylene glycol and diluted with water to constant volume， obtaining an injection containing arctigenin 10.0 mg/ml. The Fructus Arctii powder was obtained by crushing the Fructus Arctii to 100 mesh， and contained 0.33% arctigenin and 4.33% arctiin. The contents of arctigenin and arctiin in the Fructus Arctii powder was determined by the method recommended in the Veterinary Pharmacopoeia of the People’s Republic of China， and the dosages of Fructus Arctii powder and arctigenin for piglets were determined according to Veterinary Pharmacopoeia of the People’s Republic of China.

Instruments
　　1200 high-performance liquid chromatograph， produced by AgiLent; D3024R high-speed centrifuge， produced by SCILO-GEX; electronic balance （±0.000 1 g）， produced by Sartorius， Germany; N-EVAPTM111 nitrogen blower， produced by Louis， USA.

Animals
　　Six grown piglets， half male and half male， weighing （30.0±5.0） kg， numbered according to No.1-6， were purchased from Wuhan Bomu Biotechnology Co.， Ltd. After purchase， they were raised with free drinking and eating and observed normally for 10 d， and fasted for 12 h before administration. The feed was a full-price ration without any drugs.

Methods

Chromatographic conditions
　　Chromatographic column： AgiLent SB-C18 column （250 mm×4.6 mm， 5 μm）; detection wavelength： 280 nm; mobile phase： acetonitrile-water （35∶65， V∶V）; flow rate： 1.0 ml/min; sample volume： 10 μl; column temperature： （30±0.1） ℃[15].

Plasma sample treatment
　　The plasma sample was taken out of the refrigerator at -20 ℃， and thawed naturally at room temperature. A certain amount of the plasma sample （0.5 ml） were accurately pipetted into a 5.0 ml centrifuge tube， added with 2.0 ml dichloromethane， and vortex-oscillated for 60 s. Centrifugation was performed at 10 000 r/min for 10.0 min to obtain the dichloromethane layer. The sample was extracted for 2 times in total by the same method， and the two dichloromethane layers were merged， and added in a 15.0 ml centrifuge tube and blown to dryness with nitrogen in a 40 ℃ water bath. The residue was dissolved in 200 μl of mobile phase and centrifuged at 10 000 r/min for 10.0 min. The supernatant was filtered with a 0.22 μm filter membrane， and 30 μl was injected and analyzed. The chromatogram was recorded[15].

Drawing of standard curve
　　Into seven 5.0 ml polypropylene centrifuge tubes， 500 μl of blank plasma was added， respectively. Then， small amounts of the high-concentration arctigenin standard working solution were added to the centrifuge tubes， respectively， finally obtaining plasma samples with arctigenin concentrations of 0.05， 0.10， 0.25， 0.50， 1.00， 2.50 and 5.00 μg/ml， respectively. The plasma samples were treated according to the sample treatment method established in the experiment， and then loaded for HPLC analysis， and the chromatogram and peak area of arctigenin were recorded. With the peak area （x） as the abscissa and the concentration （y） as the ordinate， a standard working curve for plasma samples was established， and a regression equation was obtained.

Methodology validation
　　Into 5.0 ml polypropylene centrifuge tubes， 500 μl of blank plasma was added， respectively， and the diluted arctigenin standard working solutions of high， medium and low concentrations were then added in sequence， respectively， followed by mixing well， giving plasma samples with arctigenin concentrations of 5.0， 1.0 and 0.1 μg/ml. The plasma samples were treated in accordance with the plasma treatment method， and HPLC detection and analysis were performed.  For each concentration， 5 parallel tests were set up in one day to investigate the intra-day coefficient of variation， and the test was repeated continuously for 5 d to determine the inter-day coefficient of variation. The recovery， accuracy， precision and sensitivity of the method were calculated.
　　The accuracy， precision and sensitivity of the method were calculated according to the following formulas：
　　Accuracy （extraction recovery） = Peak area of the injected drug after sample addition and extraction/Peak area after injection of standard solution×100%;
　　Precision （intra-day and inter-day coefficient of variation） = Standard deviation/Average×100%;
　　Limit of detection （LOD）： When the signal-to-noise ratio that can be measured by the instrument is （S/N）≥3， the lowest concentration of the drug in the sample is defined as LOD.
　　Limit of quantification （LOQ）： When the signal-to-noise ratio that can be measured by the instrument is （S/N）≥10， the lowest concentration of the drug in the sample is defined as LOQ.

Pharmacokinetic test design
　　The piglets were fasted 12.0 h before administration， and weighed， and the anterior vena cava blood sample was collected as a blank control. Each piglet was administered by gavage with 1.0 mg/kg bw of Fructus Arctii water extract powder. After intragastric administration， about 5.0 ml of blood was collected for each piglet at 0.167， 0.333， 0.5， 0.75， 1.0， 1.5， 2.0， 3.0， 4.0， 5.0， 6.0， 8.0， 12.0， 24.0 and 48.0 h. The collected blood samples were added in polypropylene centrifuge tubes with sodium heparin， centrifuged at 3 000 r/min for 15.0 min to separate plasma. The supernatant was frozen and stored at -20 ℃ for determination[16].

Determination of drug concentration in pig plasma and data analysis
　　Plasma samples collected at different time points after administration were subjected to post-processing HPLC detection and analysis according to the processing method of plasma samples in "Plasma sample treatment". The peak areas of arctigenin were collected and substituted into the standard curve regression equation to calculate the plasma arctigenin concentration. The pharmacokinetic model and parameter calculation used the 3p97 software.

Results and Analysis

Chromatographic behavior of arctigenin in plasma
　　The retention time of arctigenin was 14.0 min， the peak shape was good， and the baseline was stable. The chromatogram of blank plasma is shown in Fig. 1. The chromatogram of blank plasma with arctigenin （1.0 μg/ml） is shown in Fig. 2. The chromatogram of sample plasma （0.55 μg/ml） is shown in Fig. 3.

Standard working curve
　　The standard arctigenin had a good linear relationship in the range of 0.05-10.0 μg/ml， and the linear equation was y=0.026 5x-0.032 8， r=0.999 8. The standard working curve of arctigenin in piglet plasma is shown in Fig. 4.

Methodological validation results
　　The minimum detection limit of arctigenin in piglet plasma was 0.025 μg/ml， and the minimum quantification limit was 0.05 μg/ml. Arctigenin had a good linear relationship in the range of 0.05-5.0 μg/ml， and the regression equation was y=0.026 4x-0.029 5， r=0.999 8. The recovery of this method was greater than 83.35%， the intra-day coefficient of variation was within 3.51%， and the inter-day coefficient of variation was within 5.90%. The recovery of arctigenin in piglet blank plasma is shown in Table 1， and the coefficient of variation is shown in Table 2.

Pharmacokinetic test data
　　After the administration of a single dose of Fructus Arctii water extract powder （1.0 g/kg bw）， the blood concentration in piglets is shown in Table 3， the main pharmacokinetic parameters are shown in Table 4， and the blood concentration-time curve is shown in Fig. 5.
　　 After oral administration of the Fructus Arctii water extract， the blood concentration-time data of arctigenin in piglets conformed to a two-compartment model with absorption. The drug-time curve showed double peaks， and the time to peak （tmax） was （0.251±0.011） h; the peak concentration （Cmax） was （0.560±0.063） μg/ml; the absorption half-life （t1/2ka） was （0.098±0.006） h; the distribution half-life （t1/2α） was 0.208±0.009 h; the elimination half-life （t1/2β） was （23.816±2.151） h; and the apparent volume of distribution （Vd） was （32.212±4.033） L/kg， indicating that the Fructus Arctii water extract was absorbed quickly in piglets， distributed widely， and metabolically eliminated extremely slowly.

Conclusions and Discussion
　　After intragastric administration of the Fructus Arctii water extract， the absorption half-life of arctigenin in piglets t1/2ka （0.098±0.006） h was shorter， and the time to peak tmax （0.251±0.011） h was faster， indicating that its absorption was rapid， and it took a short time to reach the highest blood concentration. The elimination half-life t1/2β （23.816±2.151） h was long， the apparent volume of distribution Vd （32.212±4.033） L/kg was large， and the peak concentration cmax （0.560±0.063） μg/ml was relatively low， indicating that arctigenin was eliminated slowly in piglets and distributed to the tissues to a greater extent， resulting in a relatively low blood concentration in the blood and a longer action time in piglets. The arctigenin reported by Li[17] showed strong absorption capacity in rats and beagle dogs， and distributed rapidly and widely in the tissues of rats， reaching a peak and width at 2.5 h （could be detected in almost all tissues and organs）， and the concentration of arctigenin in the intestine was the highest， followed by the heart， liver， pancreas and kidney. It is similar to the results of this study that the water extract of Fructus Arctii was absorbed rapidly and widely distributed in piglets after intragastric administration.
　　The blood concentration-time curve of the Fructus Arctii water extract after oral administration showed obvious double peaks. The first peak time （Tmax） of the drug was 0.5 h， and the peak concentration （Cmax） was （0.796±0.022） μg/ml. The second peak was reached at 4.0 h， and the peak concentration （Cmax） was （0.540±0.018） μg/ml. It has certain differences from the study reported by Cai[18] that after oral administration of different doses of arctigenin and its valine ester derivatives in Wistar rats， the plasma concentration-time curve of arctigenin conformed to the characteristics of a one-compartment model， and the absorption after oral administration was a nonlinear kinetic process， and there might be a saturation process. It may be caused by differences in animal species. Gao[19] reported that arctigenin had extensive glucuronic acid hydrolysis in the liver， intestines and plasma， which might be related to the double absorption peaks of the drug. The existence of double absorption peaks further proved that its retention time in the body was longer. Ikeda[20] reported that the pharmacokinetics of arctigenin in humans was a non-linear model， and a second peak was observed in the absorption of arctiin and arctigenin， suggesting an intrahepatic circulation. Gao[21] reckoned that glucuronidation is the main intestinal and liver metabolic pathway of arctigenin， and the glucuronide of arctigenin excreted in the bile can be further hydrolyzed back to the parent compound， indicating its potential enterohepatic circulation. These research reports are the same as the double absorption peaks of arctigenin in piglets after the administration of the Fructus Arctii water extract， which provides further support for the hepatoenteral circulation of arctigenin in piglets. 　　Zeng[22] found that the function of P-glycoprotein in diabetic rats was impaired. Arctigenin is a P-glycoprotein substrate， which caused the Cmax and AUC0-10 h values in diabetic rats after oral administration of arctigenin increased by 356.8% and 223.4%， respectively， compared with normal rats， but after intravenous injection， there was no significant difference in Cmax and AUC0-10 h values of arctigenin between normal and diabetic rats. Before and during the experiment in this study， it was observed that the physiological state of the piglets was not affected. There was no obvious difference in blood drug concentration and pharmacokinetic parameters of piglets of different sexes. Combined with Zeng’s research report， it can be inferred that the pharmacological parameters of arctigenin after oral administration under pathological conditions of piglets will not differ much from the pharmacokinetic parameters under healthy conditions， but the actual situation needs further research to confirm.
　　In this study， HLPC was used to establish a quantitative analysis method for arctigenin in piglet plasma， and to study the pharmacokinetic characteristics of arctigenin in the water extract of Fructus Arctii in piglets. After oral administration of the water extract powder of Fructus Arctii， the blood concentration-time data of arctigenin conformed to a two-compartment model with absorption in piglets， with the characteristics of fast absorption， wide distribution and slow elimination. Arctigenin might have a hepatoenteral circulation in piglets， and the drug effect could last for a long time. It is recommended to reduce the dosage and frequency of medication when used clinically to prevent side effects caused by the accumulation of the drug in the body.

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