The use of turmeric as a spice and a medicine is very popular in Asia. The pharmacological activities of its major compound, curcumin, have been studied intensively in laboratories all over the world.3,11,37
The poor bioavailability of curcumin also drew many scientists' attention, and enhancing bioavailability has been the objective of several studies.31,34,38
In the present study, the transport of curcumin was investigated using the in vitro
Caco-2 cell monolayer model. A simple hypothesis was verified in this well-established and widely used model. We hypothesized that the lipophilic components (e.g
., turmerones) in turmeric extract would affect the transport of curcumin in the intestine. Therefore the effects of turmerones (isolated from turmeric crude extract) on curcumin transport were explored in the present study.
Our results demonstrated that in the presence of turmerones, amounts of curcumin transported from the apical side to the basolateral side (absorption) of Caco-2 cell monolayers were relatively higher than those without turmerones. At the same time, the amounts of curcumin inside the Caco-2 cells were significantly higher when turmerones were present in the transport system (), especially when AL was present (). The in vitro
transport of curcumin with turmerones was reported here for the first time. By increasing the DMSO concentration from 1% to 5% (vol/vol) in the transport buffer, more curcumin was available for the in vitro
transport experiment. As a result, the curcumin amount in the sample solution collected on the basolateral side of the Caco-2 cell monolayer could be analyzed by HPLC, even though the concentration was low. Besides, because intestinal solubility may be far greater than the solubility measured in water, the use of aqueous solubility to predict drug absorption can therefore lead to very pronounced underestimates of the oral bioavailability.39
Hence, DMSO was used to increase solubility of curcumin, and this method enabled the evaluation of curcumin transport in the Caco-2 model with other substances (e.g
., turmerones in this study). The increased DMSO concentration did not affect the viability of Caco-2 cells after a 4-hour incubation () or the integrity (reflected by TEER) of the monolayers, and it was a more feasible method to increase the curcumin solubility than alternative heat treatment or addition of alkaline in transport buffer, which were used in previous studies.37,40
One of the limitations of the present experiment was that the amounts of curcumin in the sample solution (0.5
mL) collected on the basolateral side at 30, 60, or 90 minutess were too low to be analyzed by HPLC (data not shown). Instead, the total amount of curcumin in the basolateral solution (2.6
mL) was collected and analyzed. Therefore, the apparent permeability coefficient (Papp
) of curcumin could not be calculated.
As the improvement of curcumin transport in Caco-2 cell monolayers by adding tumerones was demonstrated, the effects of turmerones on intestinal cell transporter activities were also assessed. Because the yield percentages of AL and AR from turmeric extracts were 0.03% (wt/wt) and 0.027% (wt/wt), respectively,16
equal amounts of turmerones were combined or tested separately. AL was shown to inhibit P-gp activity as it inhibited the efflux and enhanced uptake of rhodamine-123 (). It also inhibited the B→A transport (efflux) of another P-gp substrate, digoxin, and decreased the net efflux ratio (). The effects were similar and comparable to those of the P-gp inhibitor verapamil (100
). As mentioned before, the presence of AL increased the amount of curcumin inside the cells, which may due to the inhibitory effects of AL toward P-gp and other transporters so that the efflux of absorbed curcumin has been decreased. In contrast, AR increased the efflux of rhodamine-123 and digoxin. When the two turmerones combined in equal amounts, the efflux of rhodamine-123 was slightly increased, whereas that of digoxin was decreased. It is possible that these two turmerones also act on other intestinal tranporters, apart from P-gp. The effect of AR dominated in rhodamine-123 transport so the combined effect was similar to that of AR alone. Conversely, AL affected digoxin transport dominantly, and AR had no significant effect; therefore, the combined effect was similar to that of AL alone. On the other hand, a simple HPLC detection method for digoxin was established in this study. In the past, [3
H]digoxin was commonly used for P-gp activity evaluation.29,30
With our HPLC detection method for digoxin, no radioactive samples need to be handled in the future, and the experimental procedures can be simplified for the transport studies using digoxin.
Previous studies have demonstrated that the methanolic extract of Curcuma
significantly increased the activity of P-gp by up-regulating the expressions of P-gp protein and MDR1
mRNA levels and that curcumin inhibited the activity of P-gp.30
A recent study also showed that the hydroethanolic extracts of Curcuma
species decreases the rhodamine-123 efflux (i.e
., P-gp activity).32
Our data presented here could explain the contradictory results of these studies. Our results also showed that AR could up-regulate the MDR1
expressions of Caco-2 cells. Unlike other studies in which the changes in gene expression induced by Curcuma
extracts or curcumin were observed after a 72-hour exposure,30,33
the changes were observed at a much earlier time in our study because the cells were exposed to curcumin or turmerones for 24 hours only. Furthermore, the changes in P-gp activities, in terms of rhodamine-123 and digoxin transport, were observed in cell monolayers exposed to turmerones for 150 minutes (a 30-minute preincubation plus the 120-minute transport experiment). The transient effect on P-gp activities was evaluated in the present study. Nevertheless, the changes of expressions of the above-mentioned genes have also been investigated using cells exposed to turmerones for 8 and 16 hours. However, no significant change has been observed (data not shown). To further elucidate the effects of turmerones on the activities of ATP-binding cassette transporters or other transporters that may relate to the transport of curcumin, future experiments should be performed using different transporter inhibitors or receptor antagonists.
Previous epidemiological studies suggested that turmeric contributes to the lower incidence of large-bowel cancers in Indians.3–5
Previous clinical studies have also demonstrated the chemopreventive effect of curcumin during the promotion/progression stages of colon cancer.21,41
In a recent clinical trial, colorectal cancer patients ingested curcumin, and both normal and malignant colorectal tissues were found to have taken up curcumin.42
Our in vitro
results demonstrated that the presence of turmerones increased the accumulation of curcumin inside intestinal epithelial cells. Our results agree with a recent clinical study in which curcumin with the noncurcuminoid components of turmeric (Biocurcumax™, Arjuna Natural Extracts, Alwaye, India) was taken by human volunteers;43
the bioavailability of Biocurcumax was sixfold higher than that of normal curcumin. Nonetheless, our study also demonstrated the roles of individual turmerones in curcumin transport. As previous studies suggested that most of the oral curcumin was excreted in the feces,11
it is possible that curcumin could reach the colon without being metabolized and that more curcumin could be absorbed into the cells with turmerones. As a result, curcumin could take advantage of its antitumor and anti-inflammatory properties toward the colorectal malignant cells. Furthermore, AR has been demonstrated to have immunostimulatory effects in human peripheral mononuclear cells in our previous study.16
Such effects might have contributed to the modulation of intestinal immunity, and the antitumor effects of curcumin might be enhanced. On the other hand, AL could have significantly inhibited the P-gp activities as shown in rhodamine-123 and digoxin transport studies ( and ). The potency of inhibition by AL (50
μg/mL represents approximately 229
) was comparable to that of the well-known P-gp inhibitor verapamil (100
). The anti–drug resistance capabilities of AL will be further investigated in multidrug-resistance cancer cells, in which the P-gp expression is significantly higher. Last but not least, the bioavailability and the local tissue accumulation of curcumin should be evaluated with turmerones in animal models so that the pharmacokinetics of these compounds could be revealed. These data will certainly provide a good foundation to future clinical trials using turmeric extracts.
In conclusion, the transport of curcumin in Caco-2 cell monolayers could be enhanced in the presence of turmerones, which were isolated from turmeric crude extract. The two turmerones showed opposite effects on P-gp activities: AL inhibited the P-gp activities, whereas AR enhanced P-gp activities as well as up-regulated MDR1, MRP2 and BCRP expressions in Caco-2 cells. These findings supported the use of turmeric extract (including curcumin and turmerones), other than curcumin alone in cancer patients, especially those with colorectal cancers.