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Effects of Curcuma spp. on P-glycoprotein function
ARTICLE IN PRESS Phytomedicine 17 (2010) 506–512
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Effects of Curcuma spp. on P-glycoprotein function Chadarat Ampasavate a, Uthai Sotanaphun b, Panadda Phattanawasin c, Nusara Piyapolrungroj d, a
Department of Pharmaceutical Sciences , Faculty of Pharmacy, Chiang Mai University, Chiang Mai, Thailand Department of Pharmacognosy, Silpakorn University, Nakhon Pathom, Thailand Department of Pharmaceutical Chemistry, Silpakorn University, Nakhon Pathom, Thailand d Department of Biopharmacy, Faculty of Pharmacy, Silpakorn University, Nakhon Pathom, Thailand b c
a r t i c l e in f o
Keywords: Curcuma longa Curcuma sp. ‘‘Khamin-oi’’ Curcuminoids P-glycoprotein
a b s t r a c t The effects of Curcuma longa (khamin chan) and Curcuma sp. ‘‘khamin-oi’’ (khamin-oi), as well as isolated major curcuminoids on intestinal P-gp functions were evaluated in vitro. The accumulation of R123 in Caco-2 cells was increased and the R123 efflux ratios were significantly decreased by both Curcuma longa and Curcuma sp. ‘‘khamin-oi’’ extracts, indicating their roles on efflux transporters. The a-b transport of daunorubicin was increased by curcumin, demethoxycurcumin and bisdemethoxycurcumin while the b-a transport was significantly decreased by curcumin and demethoxycurcumin. However, calcein-AM uptake into the human P-gp overexpression cell line, LLC-GA5-COL300, was increased by curcumin and demethoxycurcumin in a concentration-dependent manner but not affected by bisdemethoxycurcumin. These results show that curcumin and demethoxycurcumin could inhibit P-gp but bisdemethoxycurcumin may modulate the function of other efflux transporters such as MRP. Taken together, the information may indicate the impact of Curcuma longa and Curcuma sp. ‘‘khamin-oi’’ on pharmacokinetics of orally administered drugs that are P-gp substrates. & 2009 Elsevier GmbH. All rights reserved.
Introduction The role of multidrug resistance (MDR), particularly P-glycoprotein (P-gp), has been extensively studied in both cancer research and pharmaceutical sciences fields. P-gp is a 170-kDa membrane protein belonging to the ATP-binding cassette (ABC) transporter superfamily. This efflux pump extrudes its substrates from a cell in an ATP-dependent manner, resulting in decreased intracellular substrate accumulation (Schinkel and Jonker 2003). P-gp acts as a multidrug resistance (MDR) factor in tumor cells by transporting certain anticancer agents out of the cell. P-gp exists not only in tumor cells, but also in the plasma membrane of many normal tissues, especially in the brain, liver, kidney, and intestine, where it effluxes xenobiotics and interferes with drug absorption (Thiebaut et al. 1987, Gatmaitan and Arias 1993). Therefore, the modulation of P-gp may modify the pharmacokinetics of drugs. Intestinal P-gp plays an important role in limiting absorption of xenobiotics so it contributes to the low bioavailability of a variety of structurally diverse drugs such as cytotoxic drugs, cyclosporine, HIV protease inhibitors, and etc. (Schinkel and Jonker 2003). Hence, avoidance of the efflux in the intestine by the use of P-gp inhibitors might decrease the necessary dose of such
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E-mail addresses: [email protected], [email protected] (N. Piyapolrungroj). 0944-7113/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2009.09.004
drugs, and therefore the cost of treatment. Recently, it has been found that a number of dietary substances could modulate P-gp function (Zhang and Morris 2003, Honda et al. 2004, Konishi et al. 2004), suggesting that they could potentially cause food-drug interaction. Food-drug interactions, including those between herbs and drugs, have become a major concern in recent years. In Thailand, rhizomes of Curcuma longa (khamin chan) and Curcuma sp. ‘‘Khamin-oi’’ (khamin-oi) are widely used as spices in food and mostly utilized in some Thai traditional medicines. The major curcuminoids, isolated from both Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’, are curcumin, demethoxycurcumin and bisdemethoxycurcumin (Jirawongse 1995, 2004). Previously, Anuchapreeda et al. (2002) and Limtrakul et al. (2004) showed that natural curcuminoids modulated MDR1 expression and P-gp function in multidrug resistant human cervical carcinoma cell line. It is speculated that Curcuma ingestion may modified the bioavailability of coadministered P-gp substrate drugs. In 2007, Zhang et al. demonstrated that curcumin modulated P-gp expression in rat organs and changed the pharmacokinetic profile of peroral celiprolol, a P-gp substrate. Zhang and Lim (2008) showed that curcumin possessed the highest P-gp inhibitory activity among 8 components from 6 commonly consumed spices when P-gp-mediated [3H]-digoxin transport was evaluated in L-MDR1 (LLC-PK1 cells transfected with human MDR1 gene) and Caco-2 cells. Interestingly, Hou et al. (2008) showed the opposite effects on P-gp regulation and function of Curcuma methanolic extracts and curcumin in Caco-2-cells. Previous reports usually
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used the commercially available curcumin standard which includes all three curcuminoids. However, other major curcuminoids including demethoxycurcumin and bisdemethoxycurcumin has not been investigated for their intestinal P-gp modulating activity. This study was conducted to investigate P-gp modulating effects of extracts prepared from rhizomes of Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’, and the roles of major curcuminoids, including curcumin, demethoxycurcumin and bisdemethoxycurcumin, on reversing P-gp function were also evaluated. Caco-2 was used as an in vitro intestinal model to study the effects on efflux transporters. LLC-PK1 and LLC-GA5-COL300 (human P-gp overexpressed-LLC-PK1) cell lines were also utilized to confirm their roles on P-gp function.
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HPLC fingerprints of the Curcuma crude extracts The hydroethanolic extracts were diluted in methanol (100 mg/ml) before subjecting to a fingerprint analysis. Samples were analyzed using a HPLC apparatus, Hewlett Packard series 1100 (Hewlett Packard, Germany), with a UV-vis detector. The reversed-phase separation was performed in Inertsils ODS-3 (4.6 250 mm) and packed guard column Inertsils ODS-3 (4.6 50 mm). Gradient elution was performed at the flow rate of 1 ml/min with the mobile phase composed of 0.25% acetic acid and acetonitrile. Curcuminoids were detected at 425 nm for the first 0-13 min and followed by the detection at 250 nm for 13-35 min later. The retention time of each curcuminoid was compared with that of purified curcumin, demethoxycurcumin, and bisdemethoxycurcumin. HPLC peak area was used to calculate the content of isolated curcuminoid in crude extracts.
Materials and methods Materials The Caco-2 (a human colonic adenocarcinoma cell line) and LLC-PK1 (a porcine kidney epithelial cell line) were purchased from American Type Culture Collection (Rockville, MD, USA). The LLC-GA5-COL300 cell line, which was derived from a clone of LLC-PK1 cells stably transfected with a cDNA encoding the human P-gp, was obtained from the Riken Cell Bank (Ibaraki, Japan). Dulbecco’s modified Eagle medium (DMEM), Medium 199 (M199), Hanks’ balanced salt solution (HBSS), fetal bovine serum, L-glutamine, non-essential amino acids, penicillin (10,000 units/ ml)-streptomycin (10 mg/ml), and 0.25% trypsin-1 mM EDTA were obtained from Gibco BRL (Grand Island, NY, USA). Rhodamine 123 (R123), daunorubicin hydrochloride, vinblastine, verapamil, curcumin standard (95% purity), in vitro toxicity assay kit lactate dehydrogenase (LDH) based, and 0.4% trypan blue were purchased from Sigma Chemical Company (St. Louis, MO, USA). Calcein-AM was obtained from Fluka Chemie GmbH (Switzerland). Purified curcumin, demethoxycurcumin, and bisdemethoxycurcumin, kindly provided by Dr. Sotanaphun, were isolated from Curcuma longa by column chromatography. The identification of these standards was based on the analysis of 1H-NMR spectra and mass spectra in comparison with those reported (Pe ret-Almedia et al. 2005). The purity of each isolated curcuminoid ( 499%) was assessed by a TLC densitometric method in the absorbance mode at 254 nm. All other chemicals used were of reagent grade.
Cell culture Caco-2 cells passage 40-69 were grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 1% nonessential amino acids, 1% penicillin-streptomycin, and 2 mM glutamine at 37 1C in a humidified atmosphere of 5% CO2. The vinblastine-selected Caco-2 cells were cultivated in the presence of 10 nM vinblastine to induce P-gp expression (Anderle et al. 1998). The culture media were changed to a fresh medium without vinblastine 24 hr before experiments. LLC-PK1 cells were grown in M199 supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin. LLC-GA5-COL300 cell line was cultured in M199 supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillinstreptomycin, and 300 ng/ml colchicines, as previously reported (Tanigawara et al. 1992; Ueda et al. 1992). Both cell lines were maintained in a humidified atmosphere of 5% CO2 at 37 1C. The media were replaced for a fresh and colchicine-free medium 6 hr before experiments. Preparation of test compounds and cytotoxicity tests Curcuma extracts and curcuminoids were first dissolved in DMSO and kept at -20 1C in darkness until use. At the start of each experiment, all test compounds were then diluted in HBSS buffer pH 7.4 with the final DMSO concentrations not exceeding 1%. The cytotoxicity of test solutions was assessed using trypan blue dye exclusion method and LDH assay (Konishi 2003).
Preparation of crude extracts from Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’
Uptake study
Samples of Curcuma longa (khamin chan) and Curcuma sp. ‘‘Khamin-oi’’ (khamin-oi) were collected from a garden patch in Amphur Maerim, Chiang Mai Province, Thailand. The samples were identified by Ms. Wannaree Jaroensup, a herbarium curator, Faculty of Pharmacy, Chiang Mai University, Chiang Mai, Thailand. The voucher specimens were deposited at the Faculty of Pharmacy Herbarium. Rhizomes of each sample were chosen and planted in the Faculty’s Herbal Garden for identity confirmation in the next season. Samples were cleaned, cut and dried in a hot air oven (50 1C) for 3 days, then powdered. Twenty grams of powdered samples were washed with 200 ml hexane, then the residues were extracted with 200 ml hydroethanolic solvent (80% ethanol, 3x). The extracts were combined and filtered prior to evaporation to dryness with a rotary evaporator (temperature below 50 1C) and a lyophilizer. The extracts were kept in desiccated, refrigerated and light-protected containers before use.
Caco-2 cells and vinblastine-selected Caco-2 cells were seeded at 80,000 cells/cm2 onto 24-well plates. Cells used for experiments were grown for 7 days. LLC-PK1 and LLC-GA5-COL300 cells (passage 21-48) were respectively seeded onto 24-well plates at cell densities 120,000 cells/cm2 and 360,000 cells/cm2. Cells grown for 3 days were used for all studies. Cells were washed with HBSS and then preincubated for 30 min in HBSS in the absence and presence of Curcuma compounds. To start the experiments, 10 mM R123 or 1 mM calcein-AM (final concentration) was added and then incubated at 37 1C. After incubation, test solutions were removed and the cells were carefully washed with ice-cold HBSS. The cells were lyzed with 0.1% Triton X-100 and the amount of R123 or the fluorescent calcein was directly determined on a microplate reader (Fusions, Packard, USA) at an excitation wavelength and an emission wavelength of 485 nm and 535 nm, respectively.
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Transport study Caco-2 cells (80,000 cells/cm2) were cultivated on polycarbonate insert of Transwells cluster (0.45 mm pore size, Costar, Cambridge, MA, USA) for 21-22 days. The transepithelial electrical resistance (TEER) of Caco-2 monolayer grown in the Transwells system was routinely monitored before and after the experiment using Millicells-ERS system (Millipore Corporation, Bedford, MA, USA) and the monolayers with TEER values greater than 300 O cm2 were used throughout. The culture medium was removed from both sides of the monolayers and then washed with HBSS before starting experiments. The cell monolayers were preincubated for 30 min in HBSS with or without Curcuma compounds. To start the experiments, 10 mM R123 or 50 mM daunorubicin final concentrations were added to the donor side (apical side for apical to basolateral (a-b) experiment and basolateral side for basolateral to apical (b-a) experiment) and then incubated at 37 1C. Samples were withdrawn from the receiver side at 30, 60, 90, and 120 min. The amount of R123 was analyzed using HPLC with fluorescence detector. The assay condition was as follows: column, ODS Hypersil (4.6 mm i.d. x 25 cm, 5 mm Agilent Technologies); mobile phase, methanol/10 mM CTAB (1:1); flow rate, 1 ml/minute; excitation wavelength, 485 nm; emission wavelength, 550 nm. The injection volume was 20 ml. Daunorubicin was quantified by a microplate reader at an excitation wavelength and an emission wavelength of 485 nm and 535 nm, respectively. All transport experiments were performed under sink condition where the concentration of the test compound in the receiver side was less than 10% of the dose applied at all time points. Transport rates determined as effective permeability coefficients (Peff) were then calculated according to the equation: Peff ðcm=secÞ ¼ J=AC0
where J is the rate of appearance of the test compound in the receiver side determined by plotting cumulative amount of the compound permeated as a function of time, A is the surface area of Transwells inserts, and C0 is the initial concentration of the compound in the donor side. Data analysis All statistical tests were performed using SPSS. One way ANOVA and Tukey tests were performed with a significant level of 5% for all tests. All data are reported as the mean7standard deviation of at least three separate measurements.
Results Plant samples and HPLC fingerprints of the Curcuma extracts The rhizomes of Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’ are closely resembled, but the differentiation was made mainly according to the characteristics of the upper-ground and underground rhizomes as well as the color of coma bracts which were whitish for Curcuma longa and red-purple for Curcuma sp. ‘‘Khamin-oi’’. The fingerprints of hydroethanolic extracts of Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’ determined by HPLC-UV are presented in Fig. 1. Each curcuminoid was separated and their identities were compared with the retention times of purified curcumin, demethoxycurcumin, and bisdemethoxycurcumin. The presences of 3 major curcuminoids including curcumin, demethoxycurcumin and bisdemethoxycurcumin in both Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’ were confirmed with HPLC. As shown in Table 1, curcumin was the highest and bisdemethoxycurcumin was the lowest curcuminoid
Fig. 1. Fingerprints of the commercially from Sigma available curcumin standard (purity o 90%!) (1), Curcuma longa (2), and Curcuma sp. ‘‘Khamin-oi’’ crude extracts. The retention times of bisdemethoxycurcumin (BDMC), demethoxycurcumin (DMC), and curcumin were 8.341, 9.007, and 9.729 min, respectively.
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content in both extracts. As seen, the total content of curcuminoids in Curcuma longa was remarkedly higher than that of Curcuma sp. ‘‘Khamin-oi’’. Cytotoxicity of Curcuma extracts and curcuminoids Trypan blue exclusion and LDH assay were performed to evaluate the toxicity of Curcuma extracts. Incubation of Caco-2 cells with 50 mg/ml Curcuma longa extract and 40 mg/ml Curcuma sp. ‘‘Khamin-oi’’ extract, for 16-20 hr, altered cell viability to about 85% of the control (without Curcuma extracts). However, the viability of cells were considered to be unaffected by 4 h incubation. To investigate the toxicity of curcuminoids, it appeared that Caco-2, LLC-PK1 and LLC-GA5-COL300 cells exhibited good viabilities in 50 mM of curcumin, demethoxycurcumin, or bisdemethoxycurcumin. As a result, the following experiments would be performed with the highest concentration of extracts and isolated curcuminoid indicated above. Effect of Curcuma extracts on uptake and transport of R123 The effects of Curcuma hydroethanolic extracts on P-gp were screened by R123 uptake and transport. The R123 uptake was performed in comparison between wild-type and vinblastineselected Caco-2 cells. The uptake into vinblastine-selected cells was lower than that of wild-type Caco-2 (82.73716.46 pmol/mg protein versus 123.15722.49 pmol/mg protein) and the accumulation was markedly increased in the presence of verapamil, a P-gp inhibitor, suggesting a higher expression of P-gp in vinblastineselected Caco-2 cells. Fifty mg/ml Curcuma longa extract and 40 mg/ ml Curcuma sp. ‘‘Khamin-oi’’ extract, which contained approximately 17 mg/ml and 6 mg/ml total curcuminoids, were used to study the Table 1 Curcuminoid contents in Curcuma extracts (mg/mg crude extract). Samples
Curcumin
Demethoxycurcumin
Bisdemethoxycurcumin
Curcuma longa Curcuma sp. ‘‘Khamin-oi’’
222.677 0.18 96.077 0.08
57.79 7 0.02 35.707 0.04
51.247 0.12 13.987 0.00
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effects on uptake and transport of R123. As can be seen in Fig. 2, both Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’ extracts increased the amount of R123 accumulation in both wild-type and vinblastineselected Caco-2 cells in a concentration-dependent manner. The effects of Curcuma extracts on P-gp function were further investigated by measuring transepithelial transport of R123 across vinblastine-selected Caco-2 monolayers. Analysis of R123 transepithelial flux revealed a marked asymmetry, yielding a net efflux value of 10. Verapamil (100 mM) significantly reduced the efflux ratio to 0.94. Curcuma longa extract (50 mg/ml) significantly increased a-b transport and decreased b-a transport of R123. Curcuma sp. ‘‘Khamin-oi’’ extract (40 mg/ml) slightly increased a-b transport but significantly decreased b-a transport of R123. As noticed, the Curcuma extracts modestly reduced the b-a transport of R123 compared to 100 mM verapamil (Fig. 3). Effects of curcumin, demethoxycurcumin and bisdemethoxycurcumin on daunorubicin transport across Caco-2 cell monolayer To compare the potency of major curcuminoids isolated from Curcuma spp., the effects of curcuminoids on daunorubicin transport across Caco-2 cell monolayers were investigated. Transepithelial transport of daunorubicin across Caco-2 monolayers showed a marked asymmetry with a net efflux value of 8. Verapamil (100 mM) significantly increased a-b transport and decreased the b-a transport of daunorubicin providing a net efflux value of 1.12. As can be seen in Fig. 4, curcumin, demethoxycurcumin and bisdemethoxycurcumin significantly increased the transport of daunorubicin in the a-b direction. However, the b-a transport of daunorubicin was significantly decreased by curcumin and demethoxycurcumin but was not affected by bisdemethoxycurcumin. The net efflux ratio was reduced to 1.5, 1.0 and 2.0 in the presence of curcumin, demethoxycurcumin and bisdemethoxycurcumin, respectively. Effects of curcumin, demethoxycurcumin, and bisdemethoxycurcumin on calcein-AM uptake by LLC-PK1 and LLC-GA5-COL300 To clarify the role of curcuminoids on P-gp efflux transporter, the comparison of calcein-AM uptake was performed in LLC-PK1 and LLC-GA5-COL300. The calcein-AM uptake into
R123 Uptake (% of control)
400
300
control 100 µM verapamil 25 µg/ml Curcuma longa 50 µg/ml Curcuma longa 20 µg/ml Curcuma sp. "Khamin-oi" 40 µg/ml Curcuma sp. "Khamin-oi"
200
100
0 Caco-2
vinblastine-selected Caco-2
Fig. 2. Effects of Curcuma extracts on R123 uptake in Caco-2 cells. The cells were incubated with 10 mM R123 in the absence and presence of Curcuma extracts at 37 1C for 1 h 100 mM verapamil was used as a positive control.
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R123 Permeability (106cm/sec)
45 control 100 µM verapamil 50 µg/ml Curcuma longa 40 µg/ml Curcuma sp. "Khamin-oi" 30 **
**
15 *** *
*
0 apical to basolateral
basolateral to apical
Fig. 3. Transepithelial transports of R123 across Caco-2 cell monolayers in the absence and presence of Curcuma extracts. 100 mM verapamil was used as a positive control. Significant differences from the corresponding control were performed by ANOVA (***p o 0.001, **po 0.01, *p o 0.05).
Daunorubicin Permeability (106cm/sec)
80
60
control 100 µM verapamil 30 µM curcumin 30 µM demethoxycurcumin 30 µM bisdemethoxycurcumin ** **
*** ***
40
*** *** 20
***
0 apical to basolateral
basolateral to apical
Fig. 4. Effects of curcuminoids on daunorubicin transport across Caco-2 cell monolayer. 100 mM verapamil was used as a positive control. Significant differences from the corresponding control were performed by ANOVA (***p o0.001, **p o 0.01).
LLC-GA5-COL300 was about 10 times lower than that of LLC-PK1, indicating higher P-gp expression in LLC-GA5-COL300 than in LLC-PK1. Calcein-AM uptake into LLC-GA5-COL300 was markedly enhanced by 100 mM verapamil, compared with that of LLC-PK1. Curcumin and demethoxycurcumin significantly increased calcein-AM uptake in both cell lines in a concentration-dependent manner but the increment observed in LLC-GA5-COL300 was markedly higher than that of LLC-PK1. Bisdemethoxycurcumin did not alter the calcein-AM uptake in both LLC-PK1 and LLC-GA5COL300, implying that bisdemethoxycurcumin could not affect the P-gp function (Fig. 5).
Discussion The affinity to secretory P-gp in the gastrointestinal tract has been shown to be a potential source of drug-drug or food-drug
interactions with respect to the extent and velocity of absorption after oral administration. Several natural compounds derived from plants have been reported to modulate the function of P-gp (Honda et al. 2004, Choi et al. 2004, Ofer et al. 2005). Natural curcuminoids extracted from Curcuma longa was previously reported to modulate P-gp function in human cervical carcinoma cell line (Anuchapreeda et al. 2002, Limtrakul et al. 2004). Zhang et al. (2007) has demonstrated the down regulation of P-gp protein level in rat intestine but up regulation and no effect were observed in rat liver and kidney, respectively. In 2008, Hou et al.. showed opposite effects of curcumin and Curcuma drugs on the expression and function of P-gp in Caco-2 cells. Due to the increase in the interest of natural remedies with pharmacological activities especially those containing Curcuma spp., two hydroethanolic extracts of Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’, which are mostly consumed in Thailand, were evaluated in the study. Moreover, purified isolated major
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511
Calcein-AM Uptake (% of control)
3000
2500
LLC-PK 1
Curcuminoid Concentration 25 µM 35 µM 50 µM
2000
1500
1000
500 100 0
ND
Calcein-AM Uptake (% of control)
3000
2500
LLC-GA5-COL300
2000
1500
1000
500 100
ND
0 control
100 µM verapamil
curcumin
demethoxycurcumin
bisdemethoxycurcumin
Fig. 5. Effects of curcuminoids on calcein-AM uptakes by LLC-PK1 and LLC-GA5-COL300. The cells were incubated with 1 mM calcein-AM in the absence and presence of curcuminoids at 37 1C for 30 min. Verapamil was used as a positive control. ND: not determined.
curcuminoids, including curcumin, demethoxycurcumin and bisdemethoxycurcumin, were used to reveal their role on reversing P-gp function. To study the effect on P-gp, the decreased accumulation and enhanced efflux of P-gp substrates that is reversible upon treatment with the inhibitors were investigated in P-gp expressing cell lines. In this study, the fluorescence R123, calcein-AM which becomes fluorescent after the cleavage by cellular esterases producing a fluorescent calcein, and cytotoxic drug daunorubicin were utilized as P-gp substrates (Lee et al. 1994, Tang et al. 2004, Troutman and Thakker 2003, Legrand et al. 1998, Perloff et al. 2003). A P-gp inhibitor verapamil was used as a positive control (Schinkel and Jonker 2003). On the basis of significant expression of P-gp in Caco-2 cells (Gutmann et al. 1999), this cell line appears to be suitable for the investigation of intestinal P-gp dependent secretion processes which may be relevant for the intestinal absorption and bioavailability of drugs. The uptake and transport experiments were performed using Caco-2 cells and Caco-2 cells culturing in medium containing vinblastine to induce more expression of P-gp (Anderle et al. 1998). LLC-PK1 that expresses low amount of P-gp at the apical membrane of cells and LLC-GA5COL300 cell line derived by transfecting LLC-PK1 with human MDR1 cDNA, which overexpresses P-gp transporters (Tanigawara
et al. 1992; Ueda et al. 1992), were used to confirm the effects on P-gp transporter. The uptake results demonstrate that extracts from both Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’ increased the level of R123 accumulation in both Caco-2 cells and vinblastineselected Caco-2 cells in a concentration-dependent manner, suggesting the competition for cellular extrusion of R123 by components present in the Curcuma extracts (Fig. 2). The in vitro transcellular transport across Caco-2 monolayers showed a decrease in b-a transport and an increase in a-b transport of R123 in the presence of Curcuma extracts assuming to be due to changes in efflux activity in response to components in these extracts (Fig. 3). Both uptake and transport results substantiated the ability of Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’ to reverse the efflux function, particularly P-gp. To reveal the role of major curcuminoids on efflux activity, the effects of curcumin, demethoxycurcumin and bisdemethoxycurcumin on daunorubicin transport across Caco-2 cell monolayer was evaluated. A significant decrease in the efflux ratio of daunorubicin by curcumin, demethoxycurcumin, and bisdemethoxycurcumin suggested that these curcuminoids could modulate the efflux transporters expressed in Caco-2, especially P-gp (Fig. 4). To confirm their effects on P-gp using LLC-PK1 and
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LLC-GA5-COL300 cell lines, only two curcuminoids, curcumin and demethoxycurcumin, significantly enhanced intracellular accumulation of calcein-AM in a concentration-dependent manner but bisdemethoxycurcumin did not affect calcein-AM uptake (Fig. 5). As noticed, the effect of bisdemethoxycurcumin on efflux activity performed in Caco-2 cells was different from those observed in LLC-PK1 and LLC-GA5-COL300. A number of transport proteins expressed in the intestine including multidrug-resistance related proteins (MRP) and the polyspecific cation transporters (OCT), may involve in drug excretion. Previous reports have revealed that MRP are functionally expressed in Caco-2 cells and contributes to the active secretion of substrates in this cell line (Anderle et al. 1998). Moreover, daunorubicin is subject to additional efflux transport via MRP and may be used to probe both MRP and P-gp activities (Schinkel and Jonker 2003). In 2006, Chearwae et al. reported that curcuminoids effectively inhibited MRP1-mediated transport in HEK293 cells. Taken together, the effects of curcuminoids on daunorubicin transport across Caco-2 monolayer may indicate the role of curcuminoids on MRP function as well. So that bisdemethoxycurcumin did not influence calcein-AM uptake in LLC-GA5-COL300 but increased a-b transport of daunorubicin across Caco-2 monolayers may be due to the effect on MRP, not Pgp. Our results reveal that curcumin and demethoxycurcumin can inhibit P-gp while bisdemethoxycurcumin may modulate the function of MRP. However, our results are contradicting to the results obtained from Hou et al. (2008), in which the hydroethanolic extracts of Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’ exhibited function in the same direction as curcumin. The presence of curcumin and demethoxycurcumin in both extracts confirmed with HPLC could explain their effects. In conclusion, this study has shown that Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’ are capable of inhibiting the activities of human intestinal P-gp. The results also indicate that curcumin and demethoxycurcumin inhibit P-gp function. The implication is that regular consumption of Curcuma longa and Curcuma sp. ‘‘Khaminoi’’ could give rise to food/herb-drug interaction so it may be prudent to avoid consuming P-gp substrate drugs with Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’. Further clinical studies should be conducted to clarify the affects of Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’ on P-gp function in human.
Acknowledgement The authors thank Research and Development Institute, Silpakorn University, THAILAND, for the financial support. References Anderle, P., Niederer, E., Rubas, W., Hilgendorf, C., Spahn-Langguth, H., WunderliAllenspach, H., Merkle, H.P., Langguth, P., 1998. P-glycoprotein (P-gp) mediated efflux in Caco-2 cell monolayers: the influence of culturing conditions and drug exposure on P-gp expression levels. J. Pharm. Sci. 87, 757–762. Anuchapreeda, S., Leechanachai, P., Smith, M.M., Ambudkar, S.V., Limtrakul, P., 2002. Modulation of P-glycoprotein expression and function by curcumin in multidrug-resistant human KB cells. Biochem. Pharmacol. 64, 573–582. Chearwae, W., Wu, C.P., Chu, H.Y., Lee, T.R., Ambudkar, S.V., Limtrakul, P., 2006. Curcuminoids purified from turmeric powder modulate the function of human multidrug resistance protein 1 (ABCC1). Cancer Chemother. Pharmacol. 57, 376–388.
Choi, C-H., Kim, J-H., Kim, S-H., 2004. Reversal of P-glycoprotein-mediated MDR by 5,7,3’,4’,5’-pentamethoxyflavone and SAR. Biochem. Biophys. Res. Commun. 320, 672–679. Gatmaitan, Z.C., Arias, I.M., 1993. Structure and function of P-glycoprotein in normal liver and small intestine. Adv. Pharmacol. 24, 77–97. Gutmann, H., Fricker, G., Torok, M., Michael, S., Beglinger, C., Drewe, J., 1999. Evidence for ABC-transporters in Caco-2 cells modulating drug uptake. Pharm. Res. 16, 402–407. Honda, Y., Ushigome, F., Koyabu, N., Morimoto, S., Shoyama, Y., Uchiumi, T., Kuwano, M., Ohtani, H., Sawada, Y., 2004. Effects of grapefruit juice and orange juice components on P-glycoprotein- and MRP2-mediated drug efflux. Br. J. Pharmacol. 143, 856–864. Hou, X., Takahashi, K., Tanaka, K., Tougou, K., Qiu, F., Komatsu, K., Takahashi, K., Azuma, J., 2008. Curcuma drugs and curcumin regulate the expression and function of P-gp in Caco-2 cells in completely opposite ways. Int. J. Pharm. 358, 224–229. Jirawongse, V.A., 1995. Khamin chan. Thai Herbal Pharmacopoeia 1995, Supplement to Thai Herbal Pharmacopoeia, Prachachon Co. Ltd., Thailand, pp. 38–44. Jirawongse, V.A., 2004. Khamin-oi. Supplement to Thai Herbal Pharmacopoeia 1995, Supplement to Thai Herbal Pharmacopoeia, Prachachon Co. Ltd., Thailand, pp. 18–26. Konishi, T., Satsu, H., Hatsugai, Y., Aizawa, K., Inakuma, T., Nagata, S., Sakuda, S., Nagasawa, H., Shimizu, M., 2004. Inhibitory effect of a bitter melon extract on the P-glycoprotein activity in intestinal Caco-2 cells. Br. J. Pharmacol. 143, 379–387. Konishi, Y., 2003. Modulation of food-derived substances on intestinal permeability in Caco-2 cell monolayers. Biosci. Biotechnol. Biochem. 67, 2297–2299. Lee, J-S., Paull, K., Alvarez, M., Hose, C., Monks, A., Grever, M., Fojo, A.T., Bates, S.E., 1994. Rhodamine efflux patterns predict P-glycoprotein substrates in the National Cancer Institute drug screen. Mol. Pharmacol. 46, 627–638. Legrand, O., Simonin, G., Perrot, J-Y., Zittoun, R., Marie, J-P., 1998. Pgp and MRP activities using calcein-AM are prognostic factors in adult acute myeloid leukemia patients. Blood 91, 4480–4488. Limtrakul, P., Anuchapreeda, S., Buddhasukh, D., 2004. Modulation of human multidrug-resistance MDR-1 gene by natural curcuminoids. BMC Cancer 4, 1–6. Ofer, M., Wolffram, S., Koggel, A., Spahn-Langguth, H., Lannguth, P., 2005. Modulation of drug transport by selected flavonoids: involvement of P-gp and OCT?. Eur. J. Pharm. Sci. 25, 263–271. ´ Pe ret-Almedia, L., Cherubino, A.P.F., Alves, R.J., Dufosse , L., Gloria, M.B.A., 2005. Separation and determination of the physico-chemical characteristics of curcumin, demethoxycurcumin, and bisdemethoxycurcumin. Food Res. Int. 38, 1039–1044. Perloff, M.D., Stormer, E., von Moltke, L.L., Greenblatt, D.J., 2003. Rapid assessment of P-glycoprotein inhibition and induction in vitro. Pharm. Res. 20, 1177–1183. Schinkel, A.H., Jonker, J.W., 2003. Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview. Adv. Drug Deliv. Rev. 55, 3–29. Tang, F., Ouyang, H., Yang, J.Z., Borchardt, R.T., 2004. Bidirectional transport of rhodamine 123 and Hoechst 33342, fluorescence probes of the binding sites on P-glycoprotein, across MDCK-MDR1 cell monolayers. J. Pharm. Sci. 93, 1185–1194. Tanigawara, Y., Okamura, N., Hirai, M., Yasuhara, M., Ueda, K., Kioka, N., Komano, T., Hori, R., 1992. Transport of digoxin by human P-glycoprotein expressed in a porcine kidney epithelial cell line (LLC-PK1). J. Pharmacol. Exp. Ther. 263, 840–845. Thiebaut, F., Tsurno, T., Hamada, H., Gottesman, M.M., Pastan, I., Willingham, M.C., 1987. Cellular localization of the multidrug-resistance gene product Pglycoprotein in normal human tissues. Proc. Natl. Acad. Sci. USA 84, 7735–7738. Troutman, M.D., Thakker, D.R., 2003. Novel experimental parameters to quantify the modulation of absorptive and secretory transport of compounds by P-glycoprotein in cell culture models of intestinal epithelium. Pharm. Res. 20, 1210–1224. Ueda, K., Okamura, N., Hirai, M., Tanigawara, Y., Saeki, T., Kioka, N., Komano, T., Hori, R., 1992. Human P-glycoprotein transports cortisol, aldosterone, and dexamethasone, but not progesterone. J. Biol. Chem. 267, 24248–24252. Zhang, S., Morris, M.E., 2003. Effect of the flavonoids biochanin A and silymarin on the P-glycoprotein-mediated transport of digoxin and vinblastine in human intestinal Caco-2 cells. Pharm. Res. 20, 1184–1191. Zhang, W., Tan, T.M.C., Lim, L., 2007. Impact of curcumin-induced changes in Pglycoprotein and CYP3A expression on the pharmacokinetics of peroral celiprolol and midazolam in rats. Drug Metab. Dispos. 35, 110–115. Zhang, W., Lim, L., 2008. Effects of spice constituents on P-gp-mediated transport and CYP3A4-mediated metabolism in vitro. Drug Metab. Dispos. 36, 1283–1290.
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Phytomedicine journal homepage: www.elsevier.de/phymed
Effects of Curcuma spp. on P-glycoprotein function Chadarat Ampasavate a, Uthai Sotanaphun b, Panadda Phattanawasin c, Nusara Piyapolrungroj d, a
Department of Pharmaceutical Sciences , Faculty of Pharmacy, Chiang Mai University, Chiang Mai, Thailand Department of Pharmacognosy, Silpakorn University, Nakhon Pathom, Thailand Department of Pharmaceutical Chemistry, Silpakorn University, Nakhon Pathom, Thailand d Department of Biopharmacy, Faculty of Pharmacy, Silpakorn University, Nakhon Pathom, Thailand b c
a r t i c l e in f o
Keywords: Curcuma longa Curcuma sp. ‘‘Khamin-oi’’ Curcuminoids P-glycoprotein
a b s t r a c t The effects of Curcuma longa (khamin chan) and Curcuma sp. ‘‘khamin-oi’’ (khamin-oi), as well as isolated major curcuminoids on intestinal P-gp functions were evaluated in vitro. The accumulation of R123 in Caco-2 cells was increased and the R123 efflux ratios were significantly decreased by both Curcuma longa and Curcuma sp. ‘‘khamin-oi’’ extracts, indicating their roles on efflux transporters. The a-b transport of daunorubicin was increased by curcumin, demethoxycurcumin and bisdemethoxycurcumin while the b-a transport was significantly decreased by curcumin and demethoxycurcumin. However, calcein-AM uptake into the human P-gp overexpression cell line, LLC-GA5-COL300, was increased by curcumin and demethoxycurcumin in a concentration-dependent manner but not affected by bisdemethoxycurcumin. These results show that curcumin and demethoxycurcumin could inhibit P-gp but bisdemethoxycurcumin may modulate the function of other efflux transporters such as MRP. Taken together, the information may indicate the impact of Curcuma longa and Curcuma sp. ‘‘khamin-oi’’ on pharmacokinetics of orally administered drugs that are P-gp substrates. & 2009 Elsevier GmbH. All rights reserved.
Introduction The role of multidrug resistance (MDR), particularly P-glycoprotein (P-gp), has been extensively studied in both cancer research and pharmaceutical sciences fields. P-gp is a 170-kDa membrane protein belonging to the ATP-binding cassette (ABC) transporter superfamily. This efflux pump extrudes its substrates from a cell in an ATP-dependent manner, resulting in decreased intracellular substrate accumulation (Schinkel and Jonker 2003). P-gp acts as a multidrug resistance (MDR) factor in tumor cells by transporting certain anticancer agents out of the cell. P-gp exists not only in tumor cells, but also in the plasma membrane of many normal tissues, especially in the brain, liver, kidney, and intestine, where it effluxes xenobiotics and interferes with drug absorption (Thiebaut et al. 1987, Gatmaitan and Arias 1993). Therefore, the modulation of P-gp may modify the pharmacokinetics of drugs. Intestinal P-gp plays an important role in limiting absorption of xenobiotics so it contributes to the low bioavailability of a variety of structurally diverse drugs such as cytotoxic drugs, cyclosporine, HIV protease inhibitors, and etc. (Schinkel and Jonker 2003). Hence, avoidance of the efflux in the intestine by the use of P-gp inhibitors might decrease the necessary dose of such
Corresponding author. Tel.: + 6634255800; fax: + 6634255801.
E-mail addresses: [email protected], [email protected] (N. Piyapolrungroj). 0944-7113/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2009.09.004
drugs, and therefore the cost of treatment. Recently, it has been found that a number of dietary substances could modulate P-gp function (Zhang and Morris 2003, Honda et al. 2004, Konishi et al. 2004), suggesting that they could potentially cause food-drug interaction. Food-drug interactions, including those between herbs and drugs, have become a major concern in recent years. In Thailand, rhizomes of Curcuma longa (khamin chan) and Curcuma sp. ‘‘Khamin-oi’’ (khamin-oi) are widely used as spices in food and mostly utilized in some Thai traditional medicines. The major curcuminoids, isolated from both Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’, are curcumin, demethoxycurcumin and bisdemethoxycurcumin (Jirawongse 1995, 2004). Previously, Anuchapreeda et al. (2002) and Limtrakul et al. (2004) showed that natural curcuminoids modulated MDR1 expression and P-gp function in multidrug resistant human cervical carcinoma cell line. It is speculated that Curcuma ingestion may modified the bioavailability of coadministered P-gp substrate drugs. In 2007, Zhang et al. demonstrated that curcumin modulated P-gp expression in rat organs and changed the pharmacokinetic profile of peroral celiprolol, a P-gp substrate. Zhang and Lim (2008) showed that curcumin possessed the highest P-gp inhibitory activity among 8 components from 6 commonly consumed spices when P-gp-mediated [3H]-digoxin transport was evaluated in L-MDR1 (LLC-PK1 cells transfected with human MDR1 gene) and Caco-2 cells. Interestingly, Hou et al. (2008) showed the opposite effects on P-gp regulation and function of Curcuma methanolic extracts and curcumin in Caco-2-cells. Previous reports usually
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used the commercially available curcumin standard which includes all three curcuminoids. However, other major curcuminoids including demethoxycurcumin and bisdemethoxycurcumin has not been investigated for their intestinal P-gp modulating activity. This study was conducted to investigate P-gp modulating effects of extracts prepared from rhizomes of Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’, and the roles of major curcuminoids, including curcumin, demethoxycurcumin and bisdemethoxycurcumin, on reversing P-gp function were also evaluated. Caco-2 was used as an in vitro intestinal model to study the effects on efflux transporters. LLC-PK1 and LLC-GA5-COL300 (human P-gp overexpressed-LLC-PK1) cell lines were also utilized to confirm their roles on P-gp function.
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HPLC fingerprints of the Curcuma crude extracts The hydroethanolic extracts were diluted in methanol (100 mg/ml) before subjecting to a fingerprint analysis. Samples were analyzed using a HPLC apparatus, Hewlett Packard series 1100 (Hewlett Packard, Germany), with a UV-vis detector. The reversed-phase separation was performed in Inertsils ODS-3 (4.6 250 mm) and packed guard column Inertsils ODS-3 (4.6 50 mm). Gradient elution was performed at the flow rate of 1 ml/min with the mobile phase composed of 0.25% acetic acid and acetonitrile. Curcuminoids were detected at 425 nm for the first 0-13 min and followed by the detection at 250 nm for 13-35 min later. The retention time of each curcuminoid was compared with that of purified curcumin, demethoxycurcumin, and bisdemethoxycurcumin. HPLC peak area was used to calculate the content of isolated curcuminoid in crude extracts.
Materials and methods Materials The Caco-2 (a human colonic adenocarcinoma cell line) and LLC-PK1 (a porcine kidney epithelial cell line) were purchased from American Type Culture Collection (Rockville, MD, USA). The LLC-GA5-COL300 cell line, which was derived from a clone of LLC-PK1 cells stably transfected with a cDNA encoding the human P-gp, was obtained from the Riken Cell Bank (Ibaraki, Japan). Dulbecco’s modified Eagle medium (DMEM), Medium 199 (M199), Hanks’ balanced salt solution (HBSS), fetal bovine serum, L-glutamine, non-essential amino acids, penicillin (10,000 units/ ml)-streptomycin (10 mg/ml), and 0.25% trypsin-1 mM EDTA were obtained from Gibco BRL (Grand Island, NY, USA). Rhodamine 123 (R123), daunorubicin hydrochloride, vinblastine, verapamil, curcumin standard (95% purity), in vitro toxicity assay kit lactate dehydrogenase (LDH) based, and 0.4% trypan blue were purchased from Sigma Chemical Company (St. Louis, MO, USA). Calcein-AM was obtained from Fluka Chemie GmbH (Switzerland). Purified curcumin, demethoxycurcumin, and bisdemethoxycurcumin, kindly provided by Dr. Sotanaphun, were isolated from Curcuma longa by column chromatography. The identification of these standards was based on the analysis of 1H-NMR spectra and mass spectra in comparison with those reported (Pe ret-Almedia et al. 2005). The purity of each isolated curcuminoid ( 499%) was assessed by a TLC densitometric method in the absorbance mode at 254 nm. All other chemicals used were of reagent grade.
Cell culture Caco-2 cells passage 40-69 were grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 1% nonessential amino acids, 1% penicillin-streptomycin, and 2 mM glutamine at 37 1C in a humidified atmosphere of 5% CO2. The vinblastine-selected Caco-2 cells were cultivated in the presence of 10 nM vinblastine to induce P-gp expression (Anderle et al. 1998). The culture media were changed to a fresh medium without vinblastine 24 hr before experiments. LLC-PK1 cells were grown in M199 supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin. LLC-GA5-COL300 cell line was cultured in M199 supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillinstreptomycin, and 300 ng/ml colchicines, as previously reported (Tanigawara et al. 1992; Ueda et al. 1992). Both cell lines were maintained in a humidified atmosphere of 5% CO2 at 37 1C. The media were replaced for a fresh and colchicine-free medium 6 hr before experiments. Preparation of test compounds and cytotoxicity tests Curcuma extracts and curcuminoids were first dissolved in DMSO and kept at -20 1C in darkness until use. At the start of each experiment, all test compounds were then diluted in HBSS buffer pH 7.4 with the final DMSO concentrations not exceeding 1%. The cytotoxicity of test solutions was assessed using trypan blue dye exclusion method and LDH assay (Konishi 2003).
Preparation of crude extracts from Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’
Uptake study
Samples of Curcuma longa (khamin chan) and Curcuma sp. ‘‘Khamin-oi’’ (khamin-oi) were collected from a garden patch in Amphur Maerim, Chiang Mai Province, Thailand. The samples were identified by Ms. Wannaree Jaroensup, a herbarium curator, Faculty of Pharmacy, Chiang Mai University, Chiang Mai, Thailand. The voucher specimens were deposited at the Faculty of Pharmacy Herbarium. Rhizomes of each sample were chosen and planted in the Faculty’s Herbal Garden for identity confirmation in the next season. Samples were cleaned, cut and dried in a hot air oven (50 1C) for 3 days, then powdered. Twenty grams of powdered samples were washed with 200 ml hexane, then the residues were extracted with 200 ml hydroethanolic solvent (80% ethanol, 3x). The extracts were combined and filtered prior to evaporation to dryness with a rotary evaporator (temperature below 50 1C) and a lyophilizer. The extracts were kept in desiccated, refrigerated and light-protected containers before use.
Caco-2 cells and vinblastine-selected Caco-2 cells were seeded at 80,000 cells/cm2 onto 24-well plates. Cells used for experiments were grown for 7 days. LLC-PK1 and LLC-GA5-COL300 cells (passage 21-48) were respectively seeded onto 24-well plates at cell densities 120,000 cells/cm2 and 360,000 cells/cm2. Cells grown for 3 days were used for all studies. Cells were washed with HBSS and then preincubated for 30 min in HBSS in the absence and presence of Curcuma compounds. To start the experiments, 10 mM R123 or 1 mM calcein-AM (final concentration) was added and then incubated at 37 1C. After incubation, test solutions were removed and the cells were carefully washed with ice-cold HBSS. The cells were lyzed with 0.1% Triton X-100 and the amount of R123 or the fluorescent calcein was directly determined on a microplate reader (Fusions, Packard, USA) at an excitation wavelength and an emission wavelength of 485 nm and 535 nm, respectively.
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Transport study Caco-2 cells (80,000 cells/cm2) were cultivated on polycarbonate insert of Transwells cluster (0.45 mm pore size, Costar, Cambridge, MA, USA) for 21-22 days. The transepithelial electrical resistance (TEER) of Caco-2 monolayer grown in the Transwells system was routinely monitored before and after the experiment using Millicells-ERS system (Millipore Corporation, Bedford, MA, USA) and the monolayers with TEER values greater than 300 O cm2 were used throughout. The culture medium was removed from both sides of the monolayers and then washed with HBSS before starting experiments. The cell monolayers were preincubated for 30 min in HBSS with or without Curcuma compounds. To start the experiments, 10 mM R123 or 50 mM daunorubicin final concentrations were added to the donor side (apical side for apical to basolateral (a-b) experiment and basolateral side for basolateral to apical (b-a) experiment) and then incubated at 37 1C. Samples were withdrawn from the receiver side at 30, 60, 90, and 120 min. The amount of R123 was analyzed using HPLC with fluorescence detector. The assay condition was as follows: column, ODS Hypersil (4.6 mm i.d. x 25 cm, 5 mm Agilent Technologies); mobile phase, methanol/10 mM CTAB (1:1); flow rate, 1 ml/minute; excitation wavelength, 485 nm; emission wavelength, 550 nm. The injection volume was 20 ml. Daunorubicin was quantified by a microplate reader at an excitation wavelength and an emission wavelength of 485 nm and 535 nm, respectively. All transport experiments were performed under sink condition where the concentration of the test compound in the receiver side was less than 10% of the dose applied at all time points. Transport rates determined as effective permeability coefficients (Peff) were then calculated according to the equation: Peff ðcm=secÞ ¼ J=AC0
where J is the rate of appearance of the test compound in the receiver side determined by plotting cumulative amount of the compound permeated as a function of time, A is the surface area of Transwells inserts, and C0 is the initial concentration of the compound in the donor side. Data analysis All statistical tests were performed using SPSS. One way ANOVA and Tukey tests were performed with a significant level of 5% for all tests. All data are reported as the mean7standard deviation of at least three separate measurements.
Results Plant samples and HPLC fingerprints of the Curcuma extracts The rhizomes of Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’ are closely resembled, but the differentiation was made mainly according to the characteristics of the upper-ground and underground rhizomes as well as the color of coma bracts which were whitish for Curcuma longa and red-purple for Curcuma sp. ‘‘Khamin-oi’’. The fingerprints of hydroethanolic extracts of Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’ determined by HPLC-UV are presented in Fig. 1. Each curcuminoid was separated and their identities were compared with the retention times of purified curcumin, demethoxycurcumin, and bisdemethoxycurcumin. The presences of 3 major curcuminoids including curcumin, demethoxycurcumin and bisdemethoxycurcumin in both Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’ were confirmed with HPLC. As shown in Table 1, curcumin was the highest and bisdemethoxycurcumin was the lowest curcuminoid
Fig. 1. Fingerprints of the commercially from Sigma available curcumin standard (purity o 90%!) (1), Curcuma longa (2), and Curcuma sp. ‘‘Khamin-oi’’ crude extracts. The retention times of bisdemethoxycurcumin (BDMC), demethoxycurcumin (DMC), and curcumin were 8.341, 9.007, and 9.729 min, respectively.
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content in both extracts. As seen, the total content of curcuminoids in Curcuma longa was remarkedly higher than that of Curcuma sp. ‘‘Khamin-oi’’. Cytotoxicity of Curcuma extracts and curcuminoids Trypan blue exclusion and LDH assay were performed to evaluate the toxicity of Curcuma extracts. Incubation of Caco-2 cells with 50 mg/ml Curcuma longa extract and 40 mg/ml Curcuma sp. ‘‘Khamin-oi’’ extract, for 16-20 hr, altered cell viability to about 85% of the control (without Curcuma extracts). However, the viability of cells were considered to be unaffected by 4 h incubation. To investigate the toxicity of curcuminoids, it appeared that Caco-2, LLC-PK1 and LLC-GA5-COL300 cells exhibited good viabilities in 50 mM of curcumin, demethoxycurcumin, or bisdemethoxycurcumin. As a result, the following experiments would be performed with the highest concentration of extracts and isolated curcuminoid indicated above. Effect of Curcuma extracts on uptake and transport of R123 The effects of Curcuma hydroethanolic extracts on P-gp were screened by R123 uptake and transport. The R123 uptake was performed in comparison between wild-type and vinblastineselected Caco-2 cells. The uptake into vinblastine-selected cells was lower than that of wild-type Caco-2 (82.73716.46 pmol/mg protein versus 123.15722.49 pmol/mg protein) and the accumulation was markedly increased in the presence of verapamil, a P-gp inhibitor, suggesting a higher expression of P-gp in vinblastineselected Caco-2 cells. Fifty mg/ml Curcuma longa extract and 40 mg/ ml Curcuma sp. ‘‘Khamin-oi’’ extract, which contained approximately 17 mg/ml and 6 mg/ml total curcuminoids, were used to study the Table 1 Curcuminoid contents in Curcuma extracts (mg/mg crude extract). Samples
Curcumin
Demethoxycurcumin
Bisdemethoxycurcumin
Curcuma longa Curcuma sp. ‘‘Khamin-oi’’
222.677 0.18 96.077 0.08
57.79 7 0.02 35.707 0.04
51.247 0.12 13.987 0.00
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effects on uptake and transport of R123. As can be seen in Fig. 2, both Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’ extracts increased the amount of R123 accumulation in both wild-type and vinblastineselected Caco-2 cells in a concentration-dependent manner. The effects of Curcuma extracts on P-gp function were further investigated by measuring transepithelial transport of R123 across vinblastine-selected Caco-2 monolayers. Analysis of R123 transepithelial flux revealed a marked asymmetry, yielding a net efflux value of 10. Verapamil (100 mM) significantly reduced the efflux ratio to 0.94. Curcuma longa extract (50 mg/ml) significantly increased a-b transport and decreased b-a transport of R123. Curcuma sp. ‘‘Khamin-oi’’ extract (40 mg/ml) slightly increased a-b transport but significantly decreased b-a transport of R123. As noticed, the Curcuma extracts modestly reduced the b-a transport of R123 compared to 100 mM verapamil (Fig. 3). Effects of curcumin, demethoxycurcumin and bisdemethoxycurcumin on daunorubicin transport across Caco-2 cell monolayer To compare the potency of major curcuminoids isolated from Curcuma spp., the effects of curcuminoids on daunorubicin transport across Caco-2 cell monolayers were investigated. Transepithelial transport of daunorubicin across Caco-2 monolayers showed a marked asymmetry with a net efflux value of 8. Verapamil (100 mM) significantly increased a-b transport and decreased the b-a transport of daunorubicin providing a net efflux value of 1.12. As can be seen in Fig. 4, curcumin, demethoxycurcumin and bisdemethoxycurcumin significantly increased the transport of daunorubicin in the a-b direction. However, the b-a transport of daunorubicin was significantly decreased by curcumin and demethoxycurcumin but was not affected by bisdemethoxycurcumin. The net efflux ratio was reduced to 1.5, 1.0 and 2.0 in the presence of curcumin, demethoxycurcumin and bisdemethoxycurcumin, respectively. Effects of curcumin, demethoxycurcumin, and bisdemethoxycurcumin on calcein-AM uptake by LLC-PK1 and LLC-GA5-COL300 To clarify the role of curcuminoids on P-gp efflux transporter, the comparison of calcein-AM uptake was performed in LLC-PK1 and LLC-GA5-COL300. The calcein-AM uptake into
R123 Uptake (% of control)
400
300
control 100 µM verapamil 25 µg/ml Curcuma longa 50 µg/ml Curcuma longa 20 µg/ml Curcuma sp. "Khamin-oi" 40 µg/ml Curcuma sp. "Khamin-oi"
200
100
0 Caco-2
vinblastine-selected Caco-2
Fig. 2. Effects of Curcuma extracts on R123 uptake in Caco-2 cells. The cells were incubated with 10 mM R123 in the absence and presence of Curcuma extracts at 37 1C for 1 h 100 mM verapamil was used as a positive control.
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R123 Permeability (106cm/sec)
45 control 100 µM verapamil 50 µg/ml Curcuma longa 40 µg/ml Curcuma sp. "Khamin-oi" 30 **
**
15 *** *
*
0 apical to basolateral
basolateral to apical
Fig. 3. Transepithelial transports of R123 across Caco-2 cell monolayers in the absence and presence of Curcuma extracts. 100 mM verapamil was used as a positive control. Significant differences from the corresponding control were performed by ANOVA (***p o 0.001, **po 0.01, *p o 0.05).
Daunorubicin Permeability (106cm/sec)
80
60
control 100 µM verapamil 30 µM curcumin 30 µM demethoxycurcumin 30 µM bisdemethoxycurcumin ** **
*** ***
40
*** *** 20
***
0 apical to basolateral
basolateral to apical
Fig. 4. Effects of curcuminoids on daunorubicin transport across Caco-2 cell monolayer. 100 mM verapamil was used as a positive control. Significant differences from the corresponding control were performed by ANOVA (***p o0.001, **p o 0.01).
LLC-GA5-COL300 was about 10 times lower than that of LLC-PK1, indicating higher P-gp expression in LLC-GA5-COL300 than in LLC-PK1. Calcein-AM uptake into LLC-GA5-COL300 was markedly enhanced by 100 mM verapamil, compared with that of LLC-PK1. Curcumin and demethoxycurcumin significantly increased calcein-AM uptake in both cell lines in a concentration-dependent manner but the increment observed in LLC-GA5-COL300 was markedly higher than that of LLC-PK1. Bisdemethoxycurcumin did not alter the calcein-AM uptake in both LLC-PK1 and LLC-GA5COL300, implying that bisdemethoxycurcumin could not affect the P-gp function (Fig. 5).
Discussion The affinity to secretory P-gp in the gastrointestinal tract has been shown to be a potential source of drug-drug or food-drug
interactions with respect to the extent and velocity of absorption after oral administration. Several natural compounds derived from plants have been reported to modulate the function of P-gp (Honda et al. 2004, Choi et al. 2004, Ofer et al. 2005). Natural curcuminoids extracted from Curcuma longa was previously reported to modulate P-gp function in human cervical carcinoma cell line (Anuchapreeda et al. 2002, Limtrakul et al. 2004). Zhang et al. (2007) has demonstrated the down regulation of P-gp protein level in rat intestine but up regulation and no effect were observed in rat liver and kidney, respectively. In 2008, Hou et al.. showed opposite effects of curcumin and Curcuma drugs on the expression and function of P-gp in Caco-2 cells. Due to the increase in the interest of natural remedies with pharmacological activities especially those containing Curcuma spp., two hydroethanolic extracts of Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’, which are mostly consumed in Thailand, were evaluated in the study. Moreover, purified isolated major
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Fig. 5. Effects of curcuminoids on calcein-AM uptakes by LLC-PK1 and LLC-GA5-COL300. The cells were incubated with 1 mM calcein-AM in the absence and presence of curcuminoids at 37 1C for 30 min. Verapamil was used as a positive control. ND: not determined.
curcuminoids, including curcumin, demethoxycurcumin and bisdemethoxycurcumin, were used to reveal their role on reversing P-gp function. To study the effect on P-gp, the decreased accumulation and enhanced efflux of P-gp substrates that is reversible upon treatment with the inhibitors were investigated in P-gp expressing cell lines. In this study, the fluorescence R123, calcein-AM which becomes fluorescent after the cleavage by cellular esterases producing a fluorescent calcein, and cytotoxic drug daunorubicin were utilized as P-gp substrates (Lee et al. 1994, Tang et al. 2004, Troutman and Thakker 2003, Legrand et al. 1998, Perloff et al. 2003). A P-gp inhibitor verapamil was used as a positive control (Schinkel and Jonker 2003). On the basis of significant expression of P-gp in Caco-2 cells (Gutmann et al. 1999), this cell line appears to be suitable for the investigation of intestinal P-gp dependent secretion processes which may be relevant for the intestinal absorption and bioavailability of drugs. The uptake and transport experiments were performed using Caco-2 cells and Caco-2 cells culturing in medium containing vinblastine to induce more expression of P-gp (Anderle et al. 1998). LLC-PK1 that expresses low amount of P-gp at the apical membrane of cells and LLC-GA5COL300 cell line derived by transfecting LLC-PK1 with human MDR1 cDNA, which overexpresses P-gp transporters (Tanigawara
et al. 1992; Ueda et al. 1992), were used to confirm the effects on P-gp transporter. The uptake results demonstrate that extracts from both Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’ increased the level of R123 accumulation in both Caco-2 cells and vinblastineselected Caco-2 cells in a concentration-dependent manner, suggesting the competition for cellular extrusion of R123 by components present in the Curcuma extracts (Fig. 2). The in vitro transcellular transport across Caco-2 monolayers showed a decrease in b-a transport and an increase in a-b transport of R123 in the presence of Curcuma extracts assuming to be due to changes in efflux activity in response to components in these extracts (Fig. 3). Both uptake and transport results substantiated the ability of Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’ to reverse the efflux function, particularly P-gp. To reveal the role of major curcuminoids on efflux activity, the effects of curcumin, demethoxycurcumin and bisdemethoxycurcumin on daunorubicin transport across Caco-2 cell monolayer was evaluated. A significant decrease in the efflux ratio of daunorubicin by curcumin, demethoxycurcumin, and bisdemethoxycurcumin suggested that these curcuminoids could modulate the efflux transporters expressed in Caco-2, especially P-gp (Fig. 4). To confirm their effects on P-gp using LLC-PK1 and
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LLC-GA5-COL300 cell lines, only two curcuminoids, curcumin and demethoxycurcumin, significantly enhanced intracellular accumulation of calcein-AM in a concentration-dependent manner but bisdemethoxycurcumin did not affect calcein-AM uptake (Fig. 5). As noticed, the effect of bisdemethoxycurcumin on efflux activity performed in Caco-2 cells was different from those observed in LLC-PK1 and LLC-GA5-COL300. A number of transport proteins expressed in the intestine including multidrug-resistance related proteins (MRP) and the polyspecific cation transporters (OCT), may involve in drug excretion. Previous reports have revealed that MRP are functionally expressed in Caco-2 cells and contributes to the active secretion of substrates in this cell line (Anderle et al. 1998). Moreover, daunorubicin is subject to additional efflux transport via MRP and may be used to probe both MRP and P-gp activities (Schinkel and Jonker 2003). In 2006, Chearwae et al. reported that curcuminoids effectively inhibited MRP1-mediated transport in HEK293 cells. Taken together, the effects of curcuminoids on daunorubicin transport across Caco-2 monolayer may indicate the role of curcuminoids on MRP function as well. So that bisdemethoxycurcumin did not influence calcein-AM uptake in LLC-GA5-COL300 but increased a-b transport of daunorubicin across Caco-2 monolayers may be due to the effect on MRP, not Pgp. Our results reveal that curcumin and demethoxycurcumin can inhibit P-gp while bisdemethoxycurcumin may modulate the function of MRP. However, our results are contradicting to the results obtained from Hou et al. (2008), in which the hydroethanolic extracts of Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’ exhibited function in the same direction as curcumin. The presence of curcumin and demethoxycurcumin in both extracts confirmed with HPLC could explain their effects. In conclusion, this study has shown that Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’ are capable of inhibiting the activities of human intestinal P-gp. The results also indicate that curcumin and demethoxycurcumin inhibit P-gp function. The implication is that regular consumption of Curcuma longa and Curcuma sp. ‘‘Khaminoi’’ could give rise to food/herb-drug interaction so it may be prudent to avoid consuming P-gp substrate drugs with Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’. Further clinical studies should be conducted to clarify the affects of Curcuma longa and Curcuma sp. ‘‘Khamin-oi’’ on P-gp function in human.
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