Effect of piperine on the bioavailability and pharmacokinetics of emodin in rats

Journal of Pharmaceutical and Biomedical Analysis 115 (2015) 144–149

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Effect of piperine on the bioavailability and pharmacokinetics of emodin in rats Xin Di, Xin Wang, Xin Di, Youping Liu ∗ School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, China

a r t i c l e

i n f o

Article history: Received 2 April 2015 Received in revised form 16 June 2015 Accepted 18 June 2015 Available online 23 June 2015 Keywords: Emodin Piperine UDP-glucuronosyltransferases (UGTs) Inhibition Pharmacokinetics bioavailability

a b s t r a c t Emodin (1,3,8-trihydroxy-6-methylanthraquinone) has been widely used as a traditional medicine and was shown to possess a multitude of health-promoting properties in pre-clinical studies, but its bioavailability was low due to the extensive glucuronidation in liver and intestine, hindering the development of emodin as a feasible chemopreventive agent. In this study, piperine, as a bioenhancer, was used to enhance the bioavailability of emodin by inhibiting its glucuronidation. The pharmacokinetic profiles of emodin after oral administration of emodin (20 mg/kg) alone and in combination with piperine (20 mg/kg) to rats were investigated via a validated LC/MS/MS method. As the in vivo pharmacokinetic studies had indicated, the AUC and Cmax of emodin were increased significantly after piperine treatment, and the glucuronidation of emodin was markedly inhibited. Our study demonstrated that piperine significantly improved the in vivo bioavailability of emodin and the influence of piperine on the pharmacokinetics of emodin may be attributed to the inhibition of glucuronidation of emodin. Further research is needed to investigate the detailed mechanism of improved bioavailability of emodin via its combination with piperine. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Emodin (1,3,8-trihydroxy-6-methylanthraquinone), an active component from Chinese herbs including rhubarb (Rheum officinale B.), aloe (Aloe barbadensis M.), and leaf of senna (Cassia angustifolia), has been widely used in Chinese medicines (Fig. 1). Emodin has been known to have numerous pharmacological effects, including laxative, anti-allergic, anti-inflammatory and anti-diabetic activities [1–3]. In traditional Chinese medicines, emodin has been generally used in obesity-related diseases because of its the laxative property [4]. However, the recent reports have focused on its anti-cancer activities against several types of cancer cells, such as inhibiting ATP-induced macrophage death [5], mediating cytotoxicity in human non-small cell lung cancer cells and inhibiting the growth of hepatoma cells [6,7]. Emodin also has a conspicuously inhibitory influence on the migration and invasion of cancer cells in vitro studies [8]. As such, emodin is a prospective treatment for this disease. Despite its beneficial pharmacology activity, emodin has showed extremely low oral bioavailability in rabbits (<1%) and in rats (<3%) owing to its extensive glucuronidation [9–12], thus impeding its clinical applications. Therefore, in terms of clinical

∗ Corresponding author. Fax: +86 24 2390 2539. E-mail address: [email protected] (Y. Liu). http://dx.doi.org/10.1016/j.jpba.2015.06.027 0731-7085/© 2015 Elsevier B.V. All rights reserved.

practical applications, it is meaningful and urgent to improve the oral bioavailability of emodin. Piperine, is a principle bioactive compound of Piper nigrum and Piper longum (Fig. 1). It has been shown to possess antioxidant properties as well as antihypertensive and hepatic protective effects [13,14]. In recent years, piperine has excited increasing interest among researchers owing to its excellent bio-enhance effect and low toxicity [15]. In several studies, piperine has been shown to be an effective bioavailability enhancer of several drugs and other pharmacological active substances, such as curcumin, resveratrol, epigallocatechin-3-gallate and theophylline, in animals and in human volunteers [15–18]. For example, it was reported that co-administration of piperine and curcumin to humans and rats could enhance the bioavailability of curcumin by 2000% and 154%, respectively [16]. Several mechanisms of piperine’s bioavailabilityenhancing effect have been postulated, including the formation of polar complexes with other compounds, and inhibition of efflux transport as well as inhibition of gut and hepatic metabolism. For example, piperine has revealed strong inhibition on hepatic UDPglucuronyltransferase activities, arylhydrocarbon hydroxylase and ethylmorphine-N-demethylation in vitro [19,20]. In this paper, the study of comparative pharmacokinetics of emodin in rats, with and without co-administration of piperine, was conducted to determine whether piperine could enhance the oral bioavailability of emodin via inhibition of its glucuronidation.

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Fig. 1. Chromatograms of emodin (1) and IS (2) in the blank plasma (A); the blank plasma spiked with emodin and IS (B); a plasma sample after oral gavage of emodin to the rat at 6 h (C), respectively.

2. Experimental 2.1. Chemicals and reagents Piperine, emodin and acacetin (internal standard, IS) were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The purity of these compounds was higher than 98%. ␤-glucuronidase (Type B1, ≥1,000,000 units/g solid from bovine liver) was purchased from Sigma–Aldrich (St. Louis, MO, USA). Acetonitrile was of HPLC grade from Merck (Darmstadt, Germany). Water distilled and deionized was prepared using a Milli-Q purification system (Millipore, Milford, MA, USA) and the other solvents were of HPLC grade. 2.2. LC–MS/MS assay The LC–MS/MS system consisted of a Shimadzu SIL-HTA autosampler, a Shimadzu LC-10ADvp pump (Kyoto, Japan) and a Thermo Finnigan TSQ Quantum Ultra triple-quadrupole mass spectrometer (San Jose, CA, USA) equipped with an ESI interface. The separation was performed on a Thermo ODS-2HYPERSIL C18 column (50 mm × 4.6 mm I.D.,5 ␮m, Thermo Scientific, Pittsburgh, PA, USA) with a Diamonsil Easy Guard C18 guard cartridge (8 mm × 4 mm I.D., 5 ␮m, Dikma, Beijing, China) at 25 ◦ C. The mobile phase consisted of 0.1% formic acid (A) and acetonitrile (B) using an isocratic elution of 70% B (v/v) at the flow rate of 0.25 mL/min. The injected sample volume was 10 ␮L. The ESI source was operated in negative ionization mode. The electro-spray voltage was set at 4.2 kV, and the capillary temperature was maintained at 320 ◦ C. Nitrogen was used as the sheath gas at a flow rate of 30 Arb and auxiliary gas with a flow rate of 5 Arb for nebulization and de-solvation. Argon was used as the collision gas (1.0 m Torr) for collision-induced dissociation. Selected reaction monitoring (SRM) was conducted by monitoring the precursor-product ion transitions of m/z 269 → 225 for emodin and m/z 283 → 268 for IS with the collision energies of 35 eV and 25 eV, respectively. 2.3. Animals and treatments Sprague-Dawley female rats (SPF, weight 200 ± 20 g, n = 12, 8–10 weeks old) were obtained from Liao Ning Chang Sheng Biotechnology Co., Ltd., (Liao Ning, China). Animal experiments were conducted in accordance with the Guidelines for Animal Experimentation of Shenyang Pharmaceutical University (Shenyang, China) and all procedures were approved by the Animal Ethics Committee of this institute. The animals were acclimated in an environmentally controlled room (22–25 ◦ C, 60% relative humidity and 12 h light/dark cycle) for at least 7 days and were housed with unlimited access to food and water, except for the 12 h before and during the experiments. Six rats were administrated a

single dose of emodin (20 mg/kg; oral gavage), while the other 6 rats were received the combination of emodin (20 mg/kg; oral gavage) and piperine (20 mg/kg; oral gavage). All rats were weighed prior to the experiment to ensure for the accurate dosing of study agents.

2.4. Plasma preparation and quantification of emodin glucuronide The rat blood samples (0.3 mL each) were collected at 0.5, 1, 2, 4, 6, 8, 12, 16, and 24 h after administration by left common carotid artery intubation. Each blood sample was centrifuged at 12000 × g for 5 min at 4 ◦ C. Plasma samples (50 ␮L) were mixed with 150 ␮L of IS solution (25 ng/mL acacetin in acetonitrile) and 25 ␮L acetonitrile (or a standard or QC solution), and placed into 1.5 mL eppendorf tubes. After vortexing for 2 min and centrifuging at 12000 × g for 5 min, a 10 ␮L aliquot of each supernatant was injected into the LC–MS/MS system. Samples with concentrations exceeding those of the highest standard (500 ng/mL) were diluted with blank rat plasma prior to analysis. For the quantification of emodin glucuronide, 100 ␮L of the plasma sample was divided into two equal parts. One part was incubated with 50 ␮L of ␤-glucuronidase (1000 unit in pH 5 acetate buffer) at 37 ◦ C for 10 min before extraction, the other was simply extracted. The following procedures were carried out according to the processes described above. The difference in peak areas of emodin obtained from the samples with or without hydrolysis was used to calculate the concentration of emodin glucuronide.

2.5. Preparation of calibration standards and quality control (QC) samples The emodin stock solution was prepared by dissolving an accurately weighed sample of emodin into acetonitrile. Due to the special property of emodin and emodin glucuronide, all samples were protected from light as much as possible during preparation and processing. The IS stock solution containing 25 ng/mL of acacetin was also prepared in the same way as described above. All of the stock solutions were stored at 4 ◦ C until analysis. Then, the stock solution was diluted into a series of standard solutions. An aliquot (25 ␮L) of each diluted solution was spiked into 50 ␮L of blank plasma to yield the concentrations ranging from 1.00 to 500 ng/mL for the calibration analysis. QC samples with low, middle and high concentrations (2.50, 25.0 and 400 ng/mL) were also prepared in the same way as described above. All solutions were stored at 4 ◦ C and used within one month after preparation.

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2.6. Method validation

3. Results and discussion

The method validation assays were performed according to the United States Food and Drug Administration (FDA) guidelines [21].

3.1. Method validation

2.6.1. Selectivity A selectivity study was designed to investigate whether endogenous constituents and other substances existing in samples would interfere with the detection of emodin and IS. Selectivity was studied by comparing the chromatograms of six different batches of rat blank plasma with the corresponding spiked plasma. 2.6.2. Linearity, LLOQ and LOD Calibration curves were prepared according to Section 2.5. The linearity of calibration curves were determined by plotting the peak area ratio of emodin to IS versus the nominal concentration of emodin with weighted (1/x2 ) least square linear regression. The lower limits of quantification (LLOQ) and limits of detection (LOD) were calculated based on signal-to-noise ratio of 10:1 and 3:1, respectively, indicating that this method was sensitive for the quantitative evaluation of emodin. 2.6.3. Precision and accuracy For the evaluation of intra-day precision and accuracy, six replicate QC samples were analyzed at three concentration levels on the same day. For the evaluation of inter-day precision and accuracy, six replicates of QC samples were analyzed at three concentration levels on three consecutive days. 2.6.4. Extraction recovery The extraction recoveries were evaluated by comparing peak areas obtained from the spiked samples before extraction with those after extraction at corresponding concentrations. The extraction recoveries of emodin and IS were determined by analyzing six replicates of QC samples at three concentration levels, respectively. 2.6.5. Matrix effect The matrix effect was evaluated by comparing the peak areas of the post-extracted spiked QC samples with those of corresponding standard solutions. The matrix effect of emodin was determined by analyzing six plasma samples at three concentration levels. The matrix effect of IS was determined using a single concentration of 25 ng/mL. 2.6.6. Stability The stability experiments were performed to evaluate the stability of emodin in rat plasma under the following conditions: short-term stability at room temperature for 24 h; long-term stability at −70 ◦ C for 30 days; three freeze–thaw (room temperature) cycles on consecutive days. All stability tests in plasma were performed by analyzing three replicates of QC samples at two concentration levels (QC1 and QC3). The determined concentrations were compared with the nominal values. 2.7. Data analysis and statistical analysis A non-compartmental model was used to calculate the pharmacokinetic parameters with DAS 2.1.1 software (Mathematical Pharmacology Professional Committee of China, Shanghai, China). Statistical comparison of mean values was performed by one-way analysis of variance (ANOVA). P value smaller than 0.05 was considered statistically significant.

3.1.1. Selectivity A selectivity study was designed to investigate whether endogenous constituents and other substances existing in samples would interfere with the detection of emodin and IS. As shown in Fig. 2, there was no interference at retention times of 1.86 min (IS) and 2.80 min (emodin). The detection of IS and emodin by SRM was highly selective with no significant interference. 3.1.2. Linearity The calibration curves calculated in the range 1–500 ng/mL were linear to analyze emodin from rat plasma. The slopes, intercepts, and correlation coefficients of the regression equations were determined by least squares linear regression using a weighting factor of 1/x2 . The typical equation for the standard curves was y = 0.0410x + 0.00427 (r = 0.9953) for emodin. Deviations were within ±15% for all regression equations. 3.1.3. Precision and accuracy The intra-day and inter-day precisions and accuracies of rat plasmas were evaluated at three QC concentrations (2.5, 25, and 400 ng/mL). The results are summarized in Table 1. At all levels, the accuracy was within 85–115% with RSD less than 15%, indicating that the method is reliable and reproducible for the determination of emodin in rat plasma. 3.1.4. Recovery The extraction recovery results of emodin and IS are summarized in Table 2. The extraction recoveries of emodin from the low, medium, and high QC samples ranged from 75.2 to 81.4% with a maximum RSD of 12.7%. Meanwhile, the extraction recoveries of IS ranged from 70.9 to 75.0% with a maximum RSD of 12.5%. The results revealed that the sample pretreatment approach employed in the present work gave reproducible recoveries for emodin and IS. 3.1.5. Stability The detailed results for the stability of emodin in rat plasma are summarized in Table 3. Emodin in rat plasma was stable in the reconstituted solution for 24 h at 20 ◦ C. The relative error (% RE) of emodin in rat plasma between the initial concentrations and the concentrations following the three freeze–thaw cycles was within ±15.0%. In addition, the processed samples were also stable at −70 ◦ C for 30 days. 3.1.6. Matrix effect The matrix effects of emodin and IS were from 86.84% to 91.35%, and 84.69%, respectively. The matrix effect on the ionization of emodin and IS was not obvious under these conditions. These results indicated that the sample preparation method was satisfactory and showed that there was little appreciable matrix effect on emodin and IS. 3.2. Pharmacokinetic study of emodin and emodin glucuronide In our study, a sensitive and robust LC/MS/MS method was developed and used successfully to study the pharmacokinetics of emodin in rats after a single oral dose of emodin and combination of emodin and piperine. The mean plasma concentration–time curves of emodin and emodin glucuronide following a single oral administration of emodin and combination of emodin and piperine are shown in Fig. 3. The main pharmacokinetic parameters of emodin and emodin glucuronide are summarized in Table 4.

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1 100%

1

100%

100%

Intens

Intens

Intens

2

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

2

0.0

0.5

1.0

1.5

Time(min)

Time(min)

A

2.0

2.5

3.0

3.5

4.0

Time(min)

B

C

Fig. 2. Mean plasma concentration-time curves of emodin (A) and emodin glucuronidation (B) following oral administration of emodin alone and combination with piperine in rats (mean ± SD, n = 6), respectively. Table 1 The intra- and inter-day precisions and accuracies of emodin in rat plasma (n = 6).

Intra-day

Inter-day

Spiked (ng/mL)

Measured (mean ± SD) (ng/mL)

2.5 25 400 2.5 25 400

2.54 27.65 390.4 2.66 28.3 391.9

± ± ± ± ± ±

0.21 0.73 25.15 0.32 2.70 21.25

Accuracy (RE%)

Precision (RSD%)

1.71 10.62 -2.14 6.40 13.14 -2.15

7.3 5.0 6.3 13.4 12.2 7.1

Table 2 The recoveries of emodin and acacetin from rat plasma (n = 6). Compound

Emodin Acacetin

Recoveries (%)

Measured (mean ± SD) RSD(%) Measured (mean ± SD) RSD(%)

2.5

25

400

76.0 ± 9.7 12.7 72.2 ± 4.3 5.9

75.2 ± 2.6 3.5 75.0 ± 9.4 12.5

81.4 ± 8.8 10.8 70.9 ± 1.1 1.5

Table 3 The results of short-term stability, long-term stability and freeze–thaw cycles of emodin in rat plasma (n = 3). Concentration (ng/mL)

Stability test (RE%)

2.5 400

Short-term stability

Long-term stability

Freeze–thaw cycle

2.6 ± 0.1 373.9 ± 15.6

2.4 ± 0.2 359 ± 8.6

2.6 ± 0.1 370.6 ± 13.3

Table 4 The pharmacokinetic parameters of emodin and emodin glucuronide following oral administration of emodin alone and combination with piperine (mean ± SD, n = 6), respectively. Parameters

t1/2 (h) AUC0−t (ng h/mL) Tmax (h) Cmax (ng/mL) CLz (L/h/kg) *

Control

In presence of piperine

Emodin

Emodin glucuronide

Emodin

3.10 ± 1.81 1007 ± 181 9.60 ± 1.15 130.6 ± 24.9 19.88 ± 4.31

3.77 ± 0.28 6141 ± 724 6.4 ± 0.74 839 ± 173 3.18 ± 0.38

4.26 ± 2234 ± 10 336.6 ± 8.64 ±

p < 0.001 control vs. piperine-treated group (t-test).

Emodin glucuronide 1.61 321* 58.8* 1.02*

4.81 ± 0.50 2298 ± 405* 6.8 ± 0.94 227.7 ± 72.3* 8.25 ± 1.26*

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A

emodin emodin+piperine

400

emodin glucuronide emodin glucuronide+piperine

1000

350

900 C o n c e n tr a tio n (n g /m L )

C o n c e n t r a t io n ( n g /m L )

B

1100

300 250 200 150 100 50

800 700 600 500 400 300 200 100

0 0

4

8

12

16

20

24

0 0

4

8

Time(h)

12

16

20

24

Time(h)

Fig. 3. Mean plasma concentration-time curves of emodin (A) and emodin glucuronidation (B) following oral administration of emodin alone and combination with piperine in rats (mean ± SD, n = 6), respectively.

The pharmacokinetic parameters of our study were similar to the reported research [11]. As the pharmacokinetic results shown in Table 4, the amount of emodin glucuronide was higher than that of emodin, suggesting that emodin is rapidly glucuronidated after oral administration, which is consistent with previous studies [11,12]. A few studies have shown that piperine could markedly inhibit UGTs [15–17]. Our study showed that piperine dramatically changed the pharmacokinetics of emodin by increasing the Cmax and AUC as well as reducing the CLz. The results (Table 4) showed that piperine increased the AUC of emodin by 221% along with an elevation in the Cmax by 258% and decreased the CLz by 230%. Compared with emodin, the pharmacokinetic modulations of emodin glucuronide were even more apparent. As shown in Table 4, the AUC and Cmax of emodin glucuronide decreased by 267% and 369%, following the CLz increased by 259%. In terms of the above results, the AUC and Cmax of emodin were significantly increased and the CLz was notably decreased in piperine co-administrated group, while the AUC and Cmax of emodin glucuronide were dramatically reduced and the CLz was significantly improved, suggesting that piperine has a clinically significant influence on the pharmacokinetic processes of emodin and emodin glucuronide, particularly on the inhibition of the glucuronidation. Previous studies have demonstrated that emodin undergoes extensive phase II metabolism, especially extensive hepatic glucuronidation in rats [11,12]. Extensive phase II metabolism and enteric recycling of emodin conjugates could explain why emodin has poor oral bioavailability in rats. The crucial metabolic pathway of emodin was apparently blocked, reducing its rate of elimination, leading in turn to an increase of its accumulation in vivo. Thus, co-administrating of emodin with piperine could enhance the oral bioavailability of emodin by inhibiting its glucuronidation. In future studies, we plan to evaluate mRNA expression in liver and intestinal tissues by reverse transcription-polymerase chain reaction (RT-PCR) analysis to further verify which isoforms of UGTs are inhibited by piperine.

4. Conclusion In the present study, we sought to determine whether the piperine could serve as a potential modulator of the bioavailability of emodin in rats. The current study showed that piperine had appreciably modulated the pharmacokinetic parameters of emodin and emodin glucuronide in vivo. The results indicated that piperine increased the Cmax and AUC of emodin as well as decreasing the AUC and Cmax of emodin glucuronide, which is evidence that piperine may reduce glucuronidation of emodin and enhance its bioavailability. However, further research is needed to investigate the detailed mechanism of the improved bioavailability of emodin via its combination with piperine. Acknowledgments The authors acknowledge financial support from Doctoral Fund of Ministry of Education of China(20092134120009), and Educational Commission of Liaoning Province, China (L2013396). References [1] J. Yi, J. Yang, R. He, F. Gao, H.R. Sang, X.M. Tang, R.D. Ye, Emodin enhances Arsenic trioxide-induced apoptosis via generation of reactive oxygen species and inhibition of survival signaling, Cancer Res. 64 (2004) 108–116. [2] J.Z. Cai, S.H. Chen, Q.W. Zhang, R. Zhuang, Y. Zhang, G.Y. Lin, Determination of emodin in rat plasma by gradientelution LC–ESI-MS and its application to pharmacokinetics, Lat. Am. J. Pharm. 32 (2013) 269–274. [3] W. Liu, Z. Zheng, X. Liu, S. Gao, L. Ye, Z. Yang, M. Hu, Z.Q. Liu, Sensitive and robust UPLC–MS/MS method to determine the gender-dependent pharmacokinetics in rats of emodin and its glucuronide, J. Pharm. Biomed. Anal. 54 (2011) 1157–1162. [4] Y. Matsuda, M. Yokohira, S. Suzuki, K. Hosokawa, K. Yamakawa, Y. Zeng, F. Ninomiya, K. Saoo, T. Kuno, K. Imaida, One-year chronic toxicity study of Aloe arborescens Miller var. natalensis Berger in Wistar Hannover rats, J. Food Chem. Toxicol. 46 (2008) 733–739. [5] L.J. Liu, J. Zou, X. Liu, L.H. Jiang, J.Y. Li, Inhibition of ATP-induced macrophage death by emodin via antagonizing P2X(7) receptor, Eur. J. Pharmacol. 640 (2010) 15–19.

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