<I>Curcuma Comosa</I>

ORIGINAL ARTICLES Department of Pharmaceutics and Therapeutics1, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan; Center of Excellence in Innovative Drug Delivery and Nanomedicine2, Mahidol University, Bangkok, Thailand Effect of Curcuma comosa extracts on the functions of peptide transporter and P-glycoprotein in intestinal epithelial cells M. KAWAMI,1 Y. Y AMADA,1 F. T OSHIMORI,1 O. ISSARACHOT,2 V. B. JUNYAPRASERT,2 R. YUMOTO1, M. TAKANO1,* Received August 31, 2016, accepted October 10, 2016 *Corresponding author: Prof. Dr. Mikihisa Takano, Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan [email protected] Pharmazie 72: 123–127 (2017) doi: 10.1691/ph.2017.6147 Curcuma comosa has been widely used as a herbal medicine in Thailand; however, it remains unclear whether C. comosa influences the absorption of drugs that are substrates for the transporters in the small intestine. In this study, we investigated the effect of C. comosa extracts on the functioning of peptide transporter 1 (PEPT1), an influx transporter, and P-glycoprotein (P-gp), an efflux transporter, in Caco-2 cells and rat intestine. In Caco-2 cells, the ethanolic extract of C. comosa (CCE) lowered the uptake of glycylsarcosine (Gly-Sar), a PEPT1 substrate, while it enhanced the uptake of rhodamine 123 (Rho123), a P-gp substrate, in a concentration- dependent manner. In addition, CCE inhibited apical-to-basal transport of Gly-Sar and basal-to-apical transport of Rho123. Furthermore, the absorption of cephalexin, another PEPT1 substrate, and the exsorption of Rho123 across the rat intestine were inhibited by CCE. Conversely, CCW, the hot water extract of C. comosa, suppresses the function of PEPT1 but not of P-gp in Caco-2 cells. These results suggest that C. comosa used as a herbal medicine in Thailand may affect the intestinal absorption of certain drugs. 1. Introduction to clarify the herb-drug interactions in order to avoid possible thera- Curcuma comosa, commonly known in Thailand as wan chak peutic failures and adverse effects (Awortwe et al. 2014). motluk, is a perennial herb belonging to the Zingiberaceae family. Thai plant extracts have been studied in search for a new class The rhizome has been traditionally used for the treatment of post- of P-gp inhibitors to overcome the multi-drug resistance of tumor partum uterine bleeding and inflammation in Thailand (Weera- cells (Patanasethanont et al. 2007a, b). In addition, we established chayaphorn et al. 2010). In addition, C. comosa has pharmaco- paclitaxel-resistant HepG2 (PR-HepG2) cells, which showed logical effects such as estrogenic, choleretic, and hypolipidemic enhanced expression and function of P-gp (Takano et al. 2009). activities (Winuthayanon et al. 2009; Piyachaturawat et al. 2000, Using PR-HepG2 cells, we demonstrated that some extracts of 2002). Thus, the demand for herbal medicines has been increasing Thai plants potentiated the cytotoxicity of anticancer drugs by in recent years in Thailand. However, herbal medicines may affect inhibiting P-gp-mediated efflux (Kawami et al. 2010). Recently, the pharmacokinetics of co-administered conventional drugs (Shi we studied the P-gp modulating effect of the ethanolic extract of and Klotz 2012). Therefore, information about herb-drug interac- C. comosa, and reported that it showed P-gp inhibitory effect and tions is critical for obtaining the optimum therapeutic outcome potentiated the sensitivity of paclitaxel-resistant cancer cells to with herbal medicines such as C. comosa. paclitaxel (Takano et al. 2014). However, it is not clear whether There is some information about the pharmacokinetic properties the extract affects the functions of PEPT1 and P-gp in the intestine. of certain ingredients in C. comosa. Su et al. (2012) demonstrated In the present study, we investigated the effect of 50% ethanolic that estrogenic diarylheptanoids in C. comosa were relatively well extract of C. comosa (CCE) on the accumulation or transepithelial absorbed from rat gastrointestinal tract and distributed into various transport of PEPT1 and P-gp substrates in Caco-2 cells. In addi- organs including brain, liver, and kidney. In addition, another tion, the functions of PEPT1 and P-gp in the rat intestine were compound, (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol, in C. evaluated using an in situ closed loop study and an in vivo exsorp- comosa increased the protein expression level of GLUT4, a facili- tion study, respectively. We also compared the effect of hot water tative glucose transporter, in skeletal muscle, resulting in enhance- extract of C. comosa (CCW) with that of CCE. ment of glucose uptake (Prasannarong et al. 2012). However, there has been little information concerning the interaction of C. comosa extract and/or its ingredients with drug transporters such as peptide 2. Investigations and results transporter 1 (PEPT1) and P-glycoprotein (P-gp) in the intestine. 2.1. Effect of CCE on the uptake and transepithelial PEPT1 is localized on the brush-border membrane of the intestinal transport of [3H] glycylsarcosine (Gly-Sar) and epithelial cells, and transports a variety of dipeptides, tripeptides, and peptide-like drugs in a proton-coupled manner (Takano et al. Rho123 in Caco-2 cells 2006). P-gp is also expressed on the brush-border membrane of the The effect of CCE on [3H]Gly-Sar and Rho123 uptake in Caco-2 intestinal epithelial cells and it recognizes various structurally and cells was examined at a concentration range of 100 to 1000 μg/mL. pharmacologically unrelated neutral and positively charged hydro- CCE reduces and enhances the uptake of [3H]Gly-Sar and Rho123, phobic compounds (Takano et al. 2006). Since oral drug administra- respectively, in a concentration-dependent manner (Fig. 1A, B). tion is the most common route of administration, the interaction of In addition, we examined the effect of 100-1000 μg/mL CCE on herbal medicines with these transporters in the intestine may alter the protein amount of Caco-2 cells. However, treatment of Caco-2 the absorption of co-administered drugs. Therefore, it is important cells with the extract for one hour did not affect the protein amount Pharmazie 72 (2017) 123 ORIGINAL ARTICLES Fig. 2: Effect of CCE on the functions of PEPT1 and P-gp in rat intestine. (A) Cepha- lexin (3 mM) with (closed column) or without (open column; control) CCE (500 μg/mL) and cefadroxil (10 mM, a PEPT1 inhibitor) was administered into the closed loop in the upper intestine. Blood sample was collected at 20 min and the plasma concentration of cephalexin was measured. (B) Intestinal exsorption clearance of Rho123 under a steady state plasma concentration in the absence (open column; control) or presence (closed column) of CCE (300 μg/mL) and verapamil (100 μM, a P-gp inhibitor) in the intestinal perfusate. Each value represents the mean ± S.E. (n=3). ** p < 0.01, significantly different from each control. Fig. 1: Effect of CCE on the uptake and transepithelial transport of [3H]Gly-Sar and Rho123 in Caco-2 cells. Caco-2 cells were incubated with [3H]Gly-Sar (A) for 15 min and Rho123 (B) for 60 min in the absence (open circle; control) or presence (closed circles) of various concentrations of CCE. Transepithelial transport of [3H]Gly-Sar (C) in the apical (A)-to-basolateral (B) direction and molecules tested had no effect on the function of PEPT1 and P-gp Rho123 (D) in B-to-A direction across Caco-2 monolayers cultured on Tran- at a concentration range from 0.1 to 10 μM. swell® in the absence (open circles; controls) or presence (closed circles) of CCE (500 μg/mL) in apical side. Each value represents the mean ± S.E. (n=3). * p < 0.05, ** p < 0.01, significantly different from each control. 2.4. Effect of CCW on the functions of PEPT1 and P-gp in Caco-2 cells and rats It is important to determine the effect of CCW, as it is generally of the cells (data not shown), indicating that the extract would not consumed orally after boiling C. comosa in water. Therefore, the have apparent cell toxicity. effect of CCW on the functions of PEPT1 and P-gp in Caco-2 cells PEPT1 is expressed at the apical side of Caco-2 cell monolayers was determined. [3H]Gly-Sar uptake in Caco-2 cells significantly (Takano et al. 2006). In addition, transepithelial transport of decreased in the presence of CCW at a concentration of 1000 μg/ Rho123 from the basolateral (B)-to-apical (A) direction across mL, but not at 100 or 500 μg/mL (Fig. 4A). However, CCW had no Caco-2 cell monolayers is much higher than that from A-to-B effect on Rho123 uptake (Fig. 4B). (Takano et al. 1998). Therefore, the effect of CCE on A-to-B trans- We further examined the effect of CCW on the intestinal absorp- port of [3H]Gly-Sar and B-to-A transport of Rho123 was examined tion of cephalexin in rats by using an in situ closed loop method. in Caco-2 cells. The A-to-B transport of [3H]Gly-Sar was inhibited CCW (500 μg/mL) suppressed the plasma concentration of cepha- by CCE (Fig. 1C). In addition, CCE administration to the apical lexin at 20 min post administration (Fig. 4C), indicating that CCW side significantly inhibited the B-to-A transport of Rho123 (Fig. could inhibit the PEPT1-mediated absorption of cephalexin from 1D). Such inhibition indicates that CCE suppresses the transepi- the intestine under in vivo conditions. thelial transport of Gly-Sar and Rho123 by inhibiting the functions of PEPT1 and P-gp in Caco-2 cells, respectively.

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