Comparative pharmacokinetics of oxyresveratrol alone and in combination with piperine as a bioenhancer in rats

Artocarpus lacucha Buch.–Ham. has several pharmacological activities, and its major bioactive component is oxyresveratrol (2,4,3′,5′–tetrahydroxystilbene, Fig. 1). This compound can inhibit tyrosinase, the major enzyme involved in melanin production in humans, which can have a skin whitening effect [1‐3]. Moreover, oxyresveratrol has many other pharmacological activities such as antioxidant [4], anti–inflammatory [5, 6] and antiviral [7, 8] effects, as well as a demonstrated neuroprotective effect in vitro [9]. Oxyresveratrol does not show mutagenic activity within the concentration range of 5–100 μg/mL [10]. Therefore, this compound might have potential for further vivo study and development as a phytopharmaceutical product for clinical use. Chen et al. [11] performed a pharmacokinetic study of oxyresveratrol in rats by oral dosing at 100–400 mg/kg. Interestingly, they reported absolute oral bioavailability of approximately 10–15%, with a rapid T_max occurring approximately 15 min after dosing. Breuer et al. [12] investigated the tissue distribution of oxyresveratrol at 40 mg/kg after intraperitoneal administration in rats. They found that this compound mainly resided in plasma, and only a minimal concentration reached the brain. Mei et al. [13, 14] reported that oxyresveratrol was biotransformed by phase ΙΙ conjugation, especially glucuronidation, with the majority of metabolites excreted in urine within 12 h of dosing.

Recently, there have been attempts to improve the oral bioavailability and tissue distribution of oxyresveratrol by physical modifications or by combination with bioenhancers. Regarding physical modifications, Sangsen et al. developed a self–microemulsifying drug delivery system (SMEDDS) of oxyresveratrol that could improve the oral bioavailability by 7.9–fold and prevented amyloid–β peptide–induced neurodegeneration in mice [15]. Interestingly, Johnson et al. [16] reported that a combination of resveratrol and piperine could enhance the pharmacokinetic profile of resveratrol, including increases in AUC and C_max by up to 229 and 1544%, respectively. Resveratrol has a similar chemical structure to oxyresveratrol [17]; therefore, it is possible that piperine could also enhance the pharmacokinetic profile of oxyresveratrol. In addition, Suresh and Srinivasan [18] reported that piperine reduces the hepatic microsomal activity of UGT in vitro. Furthermore, Atal et al. [19] found that piperine at doses of 10 and 25 mg/kg could decrease in vivo UGT activity by 36 and 55%, respectively. Piperine can also inhibit P–glycoprotein, a major efflux transporter found in the hepatobiliary system and in enterocytes [20].

This study aimed to investigate the pharmacokinetic profiles of oxyresveratrol alone and in combination with piperine in male Wistar rats. Doses of oxyresveratrol and piperine were based on their pharmacodynamic activity and bioenhancer activity, respectively [16]. Oxyresveratrol (100 mg/kg p.o. or 10 mg/kg i.v.) was administered alone or combined with piperine (10 mg/kg p.o. or 1 mg/kg i.v., respectively). The tissue kinetics of oxyresveratrol in pharmacologically relevant organs was also measured to determine the tissue to plasma ratio of the test compounds. Glucuronide metabolites were analyzed in plasma and excreta in order to determine the major routes of excretion after dosing. The pharmacokinetic information obtained from this study will be useful for appropriate strategies to develop oxyresveratrol as a phytopharmaceutical product.

All male Wistar rats that received oxyresveratrol alone or in combination with piperine showed normal physical appearance both pre–dose and post–dose (24 h). In addition, two markers of liver health, AST and ALT, also showed normal levels at pre–dose and post–dose for all test formulations. There were no statistically significant differences in these markers between rats administered oxyresveratrol alone or in combination with piperine. In relation to kidney markers, there were no significant changes between pre–dose and post–dose levels for all experimental groups, and all values were within the normal range for healthy rats (Table 1).

The mean plasma concentration–time profiles for oxyresveratrol alone and in combination with piperine are shown in Fig. 2. The combination of oxyresveratrol and piperine resulted in higher levels of plasma oxyresveratrol, especially 8–24 h after intravenous administration. Interestingly, oral gavage of the combination led to a higher level of plasma oxyresveratrol from 5 min until 24 h after dosing. The oral combination of oxyresveratrol and piperine had a significantly higher C_max and tended to have shorter T_max values for oxyresveratrol. Similarly, all AUC values for oxyresveratrol in combination groups were higher than groups administered oxyresveratrol alone (p < 0.05). The mean residence time for oxyresveratrol when combined with piperine also tended to be longer, especially when administered orally (11.66 vs. 7.25 h). The combination with piperine could increase oral bioavailability of oxyresveratrol approximately 2 fold (Table 2).

The tissue to plasma ratio (K_p) of oxyresveratrol alone and in combination with piperine is shown in Fig. 3. It was found that oxyresveratrol could reach most of the internal organs with a K_p of approximately 10–100 fold within 5 min, after which the K_p gradually declined at 1, 2 and 4 h after intravenous administration. For oral administration, a high K_p was detected in the stomach and small intestine from 5 min to 4 h. Following oral gavage, the tissue to plasma ratios of oxyresveratrol in other internal organs were approximately 1–100 fold from 5 min to 4 h. Surprisingly, the tissue to plasma ratio of oxyresveratrol in the brain was increased when administered as a combination with piperine.

The plasma concentration–time profile of oxyresveratrol glucuronide is shown in Fig. 4. The addition of piperine appeared to reduce the production of glucuronide metabolites from oxyresveratrol after intravenous dosing. The conversion ratio of oxyresveratrol was approximately 25 following intravenous dosing of oxyresveratrol alone, which was decreased to 1.11 following dosing with oxyresveratrol combined with piperine. For oral dosing, the conversion ratio of oxyresveratrol glucuronide also decreased after oral dosing of oxyresveratrol combined with piperine (Table 2). The percentage recovery of unchanged oxyresveratrol in urine was found to be approximately 5–10% of the intravenous dose. A negligible amount (< 1%) of unchanged oxyresveratrol was found in feces from 0 to 48 h after intravenous dosing. A significant amount of oxyresveratrol glucuronide, ranging from 10 to 30%, was found in urine from 0 to 48 h after intravenous administration. A minimal amount (< 1%) of oxyresveratrol glucuronide was found in feces after both oral and intravenous dosing from 0 to 48 h (Table 3).

Several studies have reported that oxyresveratrol has pharmacological activity with minimal toxicity. However, this compound poses a challenge for development into a phytopharmaceutical product due to its poor pharmacokinetic profile. This problem is a common phenomenon of lead compounds from natural resources, especially low oral bioavailability due to extensive metabolism in the gastrointestinal tract [11, 14, 24]. The goal of this study was to determine whether the pharmacokinetic profile of oxyresveratrol could be improved by combination with piperine, which acts as a bioenhancer in rats. During the study, all rats showed good tolerability to oral gavage and intravenous administration of oxyresveratrol, alone and in combination with piperine. There were no significant changes in physical appearance or signs of toxicity in rats in all experiments. Two markers of liver health, AST and ALT, as well as a marker of kidney health were found to be stable and within normal ranges at pre–dose and post–dose (24 h). This tolerability result implies that all test formulae had a good safety profile in the test animals, which is consistent with previous reports by several different researchers [12, 25, 26].

As evidenced by the plasma concentration–time profiles, intravenous administration of oxyresveratrol plus piperine led to higher levels of oxyresveratrol, especially during the elimination phase from 8 to 24 h. Intravenous dose was reduced to 10% of oral dose in order to mimic systemic exposure of oxyresveratrol and piperine in rats. Therefore, calculation of pharmacokinetic parameter had more reliability from similar systemic exposure between the two routes of administration. Oral gavage of the combination showed a similar pattern, with higher levels of oxyresveratrol during the absorption, distribution and elimination phases compared to oxyresveratrol alone. This result indicates that piperine could improve the pharmacokinetic profiles of oxyresveratrol administered both intravenously and orally. Firstly, the addition of piperine led to a significant increase in oxyresveratrol C_max and AUC, which was approximately 1.5–fold higher, with a shorter T_max from 2.08 to 1.30 h. Similarly, Johnson et al. [16] reported that piperine improved the resveratrol level, evidenced by an increase in both C_max and AUC by up to 229 and 1544%, respectively. Resveratrol is a well–known compound with a similar structure to oxyresveratrol; however, it presents pharmacokinetic problems due to its extensive phase II metabolism [27‐30]. Piperine could act as a bioenhancer through several mechanisms, such as by reducing UGT metabolism, inhibiting the efflux of P–glycoprotein, and by increasing the permeability of enterocytes [16, 20, 31‐33]. In rats administered oxyresveratrol combined with piperine, a shorter T_max and higher C_max could be clearly observed during the absorption phases. Prolonged MRT of oxyresveratrol was also detected with this combination, especially after oral dosing. Interestingly, our study showed a shorter T_max and higher C_max for oxyresveratrol in both formulae compared with other pharmacokinetic studies of oxyresveratrol. This might be due to the fact that the test formulation used in our study was a clear solution, which would be more readily absorbed than the suspensions used by other studies. The combination with piperine could increase the relative bioavailability of oxyresveratrol 173%. Our finding showed a similar result to Sangsen et al. [15] that SMEDDS could improve the relative bioavailability of oxyresveratrol by 7.9–fold. Both physical modifications by SMEDDS and an addition of bioenhancers generated a superior pharmacokinetic profile of oxyresveratrol than conventional formulation alone.

In our study, we measured the oxyresveratrol level in several internal organs after intravenous and oral administration. After intravenous administration, the test compound had a V_d of approximately 50 L/kg, which is considered to be a large distribution volume. This might be due to the molecular weight of oxyresveratrol of 224.07 Da and XLogP 2.8, which is considered a small lipophilic molecule. Therefore, the test compound could rapidly reach the internal organs within 5 min with a K_p of 10–100, except for the brain. Very low levels of oxyresveratrol were observed in the brain at 1, 2, and 4 h after intravenous administration of oxyresveratrol, suggesting that oxyresveratrol has limited deposition in brain tissue. Interestingly, combination with piperine could promote oxyresveratrol level in brain tissue especially at 1–2 h after administration. Mei et al. [14] reported that oxyresveratrol is a substrate of P–glycoprotein, an efflux transporter located at the blood–brain barrier. The minimal amount of oxyresveratrol in the brain tissue might be due to restricted xenobiotic penetration and efflux transport of oxyresveratrol from the brain tissue into the circulation. The addition of piperine could improve oxyresveratrol level in the brain tissue, especially in intravenous administration.

In a previous study, oxyresveratrol was reported to be mainly metabolized by the glucuronidation reaction [14, 22]. In our study, the conversion ratio of oxyresveratrol to glucuronide metabolites was 25.28 after intravenous administration of oxyresveratrol alone. These results demonstrate that oxyresveratrol was biotransformed into glucuronide metabolites at a high ratio. When combined with piperine, this conversion ratio was decreased to 1.11 after intravenous dosing and 0.80 after oral dosing. These results indicate that piperine reduced the glucuronide reaction of oxyresveratrol in test animals. Atal et al. [19] reported that piperine at doses of 10 and 25 mg/kg p.o. in mice could inhibit UGT activity by 36 and 55%, respectively. The major route of oxyresveratrol excretion appears to be via the urinary system. Higher percentages of unchanged oxyresveratrol and oxyresveratrol glucuronide were detected in urine from 0 to 48 h after dosing. Huang et al. [22] also reported that oxyresveratrol was readily excreted in urine from 0 to 12 h after administration, based on the fact that oxyresveratrol becomes more hydrophilic by biotransformation via metabolic conjugation to monoglucuronide oxyresveratrol. Similarly, our study found a higher percentage of oxyresveratrol glucuronide in urine than unchanged oxyresveratrol.

In conclusion, the addition of piperine enhanced some of the pharmacokinetic properties of oxyresveratrol via both intravenous and oral administration methods. Oral administration of oxyresveratrol combined with piperine was associated with increased C_max, AUC and MRT. Improvement of oxyresveratrol in brain tissue was observed after intravenous administration. Glucuronide metabolites of oxyresveratrol were significantly reduced when rats were administered oxyresveratrol plus piperine. Urinary excretion of oxyresveratrol glucuronide appears to be the major route of oxyresveratrol excretion. This pharmacokinetic data will be useful for further development of oxyresveratrol as a future phytopharmaceutical product.