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Effects of Devil's Claw (Harpagophytum procumbens) on the multidrug transporter ABCB1/P-glycoprotein.

ABSTRACT

Devil's Claw (Harpagophytum procumbens) a plant native to Southern Africa, has historically been used in traditional medicine to treat a wide range of diseases and currently is widely employed as anti-inflammatory and pain-relieving natural remedy in Europe and other parts of the world.

Aim of the study: Little is known about possible herb-drug interactions arising from effects of Devil's Claw on the major drug metabolizing enzymes or transporters. This study evaluated in vitro the effects of Devil's Claw on the multidrug transporter ABCBl/P-glycoprotein.

Materials and methods: The effects of three commercially available Devil's Claw preparations and that of pure harpagoside were studied in the human kidney (HK-2) proximal tubule cell line, constitutively expressing ABCBl/P-glycoprotein (P-gp). Pgp activity and expression were tested by the calcein-AM test and by Western blotting, respectively.

Results: Commercial preparations inhibited P-gp activity, even if to a different extent, while pure harpagoside was almost ineffective. In cells cultured for three days in the presence of Devil's Claw preparations or pure harpagoside, a dose-dependent P-gp upregulation was found. Conclusions: Our results demonstrate for the first time that Devil's Claw may interact with the multidrug transporter ABCBl/P-gp, the effect not appearing strictly related to the harpagoside relative content. Modulation of both P-gp activity and P-gp expression by Devil's Claw raise the possibility of herb-drug interactions, to be further explored in depth.

[c] 2009 Elsevier GmbH. All rights reserved.

ARTICLE INFO

Keywords:

Devil's Claw (Harpagophytum procumbens)

Harpagoside

P-glycopratein

ABCB1

HK-2 cells

Introduction

The multidrug transporter MDR-1/P-glycoprotein (P-gp ABCB1) is nowadays acknowledged as a major determinant for the acquisition of multidrug resistance (MDR) phenotype by tumoral cells during chemotherapy (Gillet et al. 2007). However, P-gp is also expressed physiologically in several epithelial/endothelial cells in normal tissues and organs, including intestine, liver, kidney, brain and placenta, where it is believed to play a protective role towards toxins and xenobiotics (Leslie et al. 2005). In these locations functional and/or expression changes of P-gp may influence ADMET of its substrate drugs (Varma et al. 2003). For this reason, the attention to P-gp has recently shifted to its impact on drug-drug interactions, and, more interestingly, to herb-drug interactions. In fact, a lot of plant-derived natural compounds, present in food or used as phytotherapeutics, are substrates/inhibitors/inducers of P-gp (Zhou et al. 2004; Hu et al. 2005; Izzo 2005; Adams et al. 2007; Marchetti et al. 2007).

However, in spite of growing concern and examples of herb-drug interactions, that may arise from modulation of P-gp, as well as from effects on CYPs isoenzymes (Pal and Mitra 2006; Williamson 2006), little research has been published in some herbal medications of widespread use, including Devil's Claw (DC).

DC is a plant native to Southern Africa, locally used for centuries to treat a variety of diseases. Today DC is widely marketed all over the world and its primary use is for all conditions able to cause inflammation and pain (Grant et al. 2007). The active components in DC are thought to be iridoid glycosides, mainly harpagoside, which are found in the secondary root (Qi et al. 2006).

While DC, besides other herbal extracts, has been reported by Unger and Frank (2004) to inhibit the activity of a series of CYPs, its interaction potential with the drug efflux transporter P-glycoprotein has not yet been evaluated.

Materials and methods

Materials

The monoclonal antibody anti P-gp, clone F4, was obtained from Sigma-Aldrich S.r.I. (Milano, Italy) and horseradish-peroxidase-conjugated anti-mouse IgG from Calbiochem (Milan, Italy). Bio-Rad Laboratories S.r.I. (Milan, Italy) was the supplier for Western blotting reagents. BM Chemiluminescence Blotting Substrate (POD) was from Roche Diagnostics GmbH (Mannheim-Germany). All other chemicals, media and cell culture reagents were purchased from Sigma-Aldrich S.r.I. (Milano, Italy).

Preparation of Devil's Claw extracts

Three commercial Harpagophytum procumbens (Devil's Claw) preparations, onwards named DC1, DC2 and DC3, standardized on harpagoside content (2, 1.2 and 1%, respectively, with suggested daily dosage of harpagoside ranging from 7 to 20 mg), were obtained directly from pharmacies in Pisa, Italy. Samples of the preparations were pulverized in a mortar and the obtained powder was weighed, directly dissolved (1:5 w/v) in dimethy-sulphoxide (DMSO) and stirred thoroughly for 24 h. The liquid dispersions were centrifuged and aliquots of the clear surnatant were stored at -20[degrees]C as stock solutions. On the day of the experiment, stock DC extracts were thawed and diluted in DMSO in order to obtain the appropriate concentrations. The final concentration of DMSO in culture medium was never greater than 0.2% (v/v).

HPLC analysis

Harpagoside content of the samples was evaluated as described by Sagare et al. (2001). Briefly, analysis was performed on a Beckman high-performance liquid chromatography system equipped with a Synergi[R] Hydro-RP HPLC column (Phenomenex) fitted with a SecurityGuard[R] C18 precolumn (Phenomenex). Mobile phase (methanol-water, 1:1) was delivered at a flow rate of 0.8 ml/min. Calibration was based on external standards using a stock solution of pure harpagoside diluted with methanol to prepare working solutions containing 1, 0.5, 0.25, 0.125, and 0.0625 mg/ml harpagoside. A System Gold 166NM detector was used to perform UV detection at 278 nm. The concentration of harpagoside in DMSO stocks extracts from DC commercial preparations used in this study, resulted to be -in triplicate measurements- 0.75[+ or -]0.04mg/ml (DC1), 2.25[+ or -]0.02 mg/ml (DC2) and 1.68[+ or -]0.08 mg/ml (DC3), respectively.

Cell culture

As a model for P-gp, we used the immortalized human proximal tubule cell line HK-2, purchased from the American Type Cell Collection and cultured as previously reported (Romiti et al. 2004). All experiments were performed on confluent cells maintained for 48 h in serum-free medium.

Western blotting analysis

Protein expression was evaluated in crude membranes of cells cultured for 72 hours in the presence or in the absence of the test compounds. Aliquots (25 [micro]g) of membrane proteins were separated by SDS-PAGE on 6% acrylamide Laemmly minigels and transferred overnight onto nitrocellulose membrane. Equal loading conditions in the gels were routinely ascertained by staining blots with Ponceau S. After blocking, membranes were incubated for 2 hrs at room temperature with primary monoclonal antibody F4, then incubated with peroxidase-conjugated secondary antibody for 60 min at room temperature. Blots were developed using chemiluminescence detection system and analysed by densitometry (Gel Documentation System Chemi Doc and Quantity One version 4.3 software, Bio-Rad, Milan, Italy).

Functional transport studies

P-gp activity was evaluated by the fluorimetric measurement of the intracellular accumulation of calcein produced by ester hydrolysis of the Pgp substrate calcein-AM in cells incubated for one hour in presence or in absence of test compounds. The assay was performed in 96 well plates multilabel reader (Wallac 1420 Perkin-Elmer Victor 3) at [lambda] exc 485 nm and [lambda] em 535 nm, respectively. The acknowledged Pgp modulator verapamil (Vp) was used as internal standard (positive control).

Cytotoxicity assay

Possible cytotoxic effects of the concentration range of DC extracts or harpagoside used throughout the experiments were screened by evaluating cell viability by the neutral red assay (TOX4 kit, Sigma) as well as by the trypan-blue exclusion test.

Modulation of esterase activity

Because in the calcein-AM assay the possible ability of test compounds to inhibit esterases might interfere with the measurement of P-gp activity, test compounds were also screened for esterase inhibition. Purified rabbit liver esterase (5U/ml) was incubated with different concentrations of test compounds and calcein-AM. After one hour the amount of free calcein was measured fluorimetrically at [lambda] exc 485 nm and [lambda] em 535 nm. Kaempferol (100 [micro]M) and PMSF (1 Mm) were used as internal controls.

Statistical analysis

Data are presented as means [+ or -] standard deviation (SD). Statistical significance was assessed using ANOVA followed by the post hoc test. Levels of p < 0.05 were considered significant.

GraphPad Prism[R] 5.0 software was used to calculate the concentrations associated with 50% inhibition of the effect ([IC.sub.50]) or 50% of the maximal response ([EC.sub.50]). using a Hill function nonlinear regression analysis.

Results

Calcein-AM assay performed on HK-2 cells exposed to extracts from different Devil's Claw commercial preparations, showed that two out of three, DC2 and DC3 ([EC.sub.50] = 303.6 and 262.4 [micro]g/ml respectively), were able to cause a small but significant increase in intracellular calcein fluorescence as compared with control (Fig. 1). Such an increase in fluorescence, induced also by the known P-gp inhibitor Verapamil, is proportional to the decrease in P-gp dependent efflux of Calcein-AM (Hollo et al. 1994) and suggests that DC extracts could contain one or more compounds able to inhibit P-gp activity. On the other hand, harpagoside, a major component of DC extracts, at concentrations ranging from 0.5 to 200 [micro]M, was unable to increase intracellular calcein fluorescence compared to controls (not shown).

[FIGURE 1 OMITTED]

Since Devil's Claw appears effective as an anti-inflammatory and analgesic agent and harpagoside has been shown to inhibit COX-2 expression (Huang et al. 2006). some NSAIDs including COX-2 selective inhibitors were also tested for potential effects on HK-2 P-gp activity. Among them, nimesulide (EC.sub.50] = 20.4 [micro]M) and even more celecoxib ([EC.sub.50] = 8.3 [micro]M), significantly increased intracellular calcein fluorescence (Fig. 2) indicating an inhibition of P-gp activity only slightly lower as compared to verapamil ([EC.sub.50] = 5.1 [micro]M). This appears by itself an interesting observation, because there are only few reports on modulation of P-gp activity by anti-inflammatory drugs (Huang et al. 2007; Angelini et al. 2008) and strongly suggests the need for further investigations on this topics.

[FIGURE 2 OMITTED]

Furthermore, because Calcein-AM test results could be influenced by changes in esterase activity, DC extracts, as well pure harpagoside were tested for a possible inhibition of this enzyme. Although esterase inhibition was not observed for any concentrations of harpagoside, all three DC extracts significantly inhibited the activity of this enzyme (Fig. 3) showing [IC.sub.50] values as 285.1, 174.9 and 156.7 [micro]g/ml for DC1, DC2 and DC3 respectively. Since modulation of esterase activity has been suggested as a new mechanism underlying particular drug interactions (Li et al. 2007), this aspect merits further attention too. Anyway, the degree of P-gp and/or esterase inhibition by DC commercial preparations was not related to the harpagoside content expected according to data reported on the label of the packaging, nor to the harpagoside concentration found in DMSO extracts of DCs, suggesting that the observed effects could be related to DC components different from harpagoside.

[FIGURE 3 OMITTED]

Our results regarding the possible effects of harpagoside or DC extracts on P-gp expression, showed a dose-dependent increase in P-gp immunoblottable amount occurring in cells cultured either in the presence of DC extracts ([EC.sub.50] = 169.5 [micro]g/ml for DC3) or in the presence of harpagoside ([EC.sub.50] = 5.2 [micro]M) (Figs. 4A, B and C). However, also in this case, no correlation was found between the content in harpagoside of the extracts and the relative increase in P-gp expression, suggesting again the involvement of other unknown chemical components present in DC preparations.

[FIGURE 4 OMITTED]

No cytotoxic effects were observed by the various concentrations of harpagoside nor by DC extracts throughout the experiments, as showed by the neutral red assay (Fig. 5) and confirmed by the trypan blue test (not shown).

[FIGURE 5 OMITTED]

Discussion

Devil's Claw (Harpagophytum procumbens) is taken by a large number of individuals suffering of chronic low back pain or other inflammatory chronic diseases, as an alternative to conventional anti-inflammatory drugs, like NSAIDs (Grant et al. 2007; Warnock et al. 2007).

DC contains a mix of substances that could be responsible for its therapeutic efficacy, in particular some iridoids (Qi et al. 2006), including harpagoside that is believed to account for much of the DC therapeutic effects (Harpagophytum procumbens, monograph 2008). Commercial preparations of DC are usually titrated on the basis of harpagoside content. This iridoid has been found to exert a series of effects possibly connected with DC anti-inflammatory properties, including inhibition of COX-2, inhibition of NF-[kappa]B activation and down-regulation of iNOS (Kaszkin et al. 2004; Huang et al. 2006; Abdelouahab and Heard 2008).

Devil's Claw is generally believed to be devoid of long-term toxicity or adverse effects and clinically important herb-drug interactions have not been described, except for a possible potentiation of warfarin action (Vlachojannis et al. 2008).

The major mediators of herb-drug interactions are the CYP450 isoenzymes and the multidrug transporter ABCB1, P-glycoprotein (Williamson 2006; Pal and Mitra 2006). While an in vitro study reported inhibitory effects of several herbal trade products including Devil's Claw on six human CYP450 expressed in the Sf9 system (Unger and Frank 2004), to our best knowledge interactions of Devil's Claw with P-gp have not been described.

For the first time we show the ability of DC and its leading iridoid compound harpagoside to interact with the multidrug transporter P-gp. In particular, DC influences both activity and expression of the transporter, while harpagoside modulates only its expression.

The model here chosen, the HK-2 cell line, constitutively expressing P-gp, has been suggested by us as a valid tool to investigate potential interactions between chemicals, including herbal drugs, with P-gp (Romiti et al. 2008; Chieli and Romiti 2008).

In this study, we found that commercial preparations of DC were able to inhibit, even to a small extent, the activity of P-gp in the calcein-AM assay. However, a possible interference on the assay could be represented by the inhibition of esterase activity by the test compound, leading to underestimation of P-gp inhibition. Interestingly, all of three commercial preparations of Devil's Claw were able to inhibit esterase activity, suggesting that the inhibitory effect on Pgp would be larger than that measured. Furthermore it has been recently shown that esterase inhibition by some grapefruit juice flavonoids, like kaempferol and others, may mediate a new drug pharmacokinetic interaction, with some ester prodrugs like lovastatin and enalapril (Li et al. 2007). We cannot exclude that DC might lead to this recently described kind of drug interaction, possibly due, at least in part, to the small amount of kaempferol present in harpagophytum roots (Betancor-Fernandez et al. 2003). Interestingly, however, pure harpagoside did not affect neither the efflux of calcein-AM, nor the activity of esterase, suggesting its failure as P-gp inhibitor. This is in agreement with the lack of correlation between the extent of observed effect of DC on P-gp activity and the harpagoside content of the preparations.

Because flavonoid aglycones, but not their respective glycosides, were able to inhibit esterase (Li et al. 2007) the glycoside moiety in the iridoid harpagoside could be the reason for its inefficiency. In this regard it should be remembered that recent extensive investigations on naturally occurring iridoids, revealing a wide range of bioactivities for these phytochemicals, show also that the aglycones are usually more active than the parent glycosides (Tundis et al. 2008). The conversion of glycosides to their aglycones is important in vivo, because it may be caused by bacteria present in intestinal flora. Anyway, other phytochemicals different from iridoids may be responsible for the DC preparations activity, e.g. flavonoids, that are well-known P-gp modulators.

Another interesting observation is that prolonged exposition to either DC extracts or pure harpagoside produced a significant, dose-dependent increase in P-gp expression. Because here too no correlation was found between DC harpagoside content and the extent of induction, the observed P-gp upregulation by Devil's Claw likely represents the overall effect of harpagoside and other unidentified constituents, eventually able to produce P-gp induction or synergizing with harpagoside (Setty and Sigal 2005). As a matter of fact, other effects induced by Devil's Claw have been shown to be dependent and not dependent on harpagoside, including downregulation of iNOS expression and inhibition of NF-[kappa]B system (Kaszkin et al. 2004).

Anyway, the observed modulation of P-gp expression is perhaps more important than the simple acute interference on Pgp-activity, because Devil's Claw is employed for chronic diseases, therefore in long-term treatments which increases the probability of undesired reactions (Pal and Mitra 2006). Interestingly, it has been estimated that during in vivo intake of extracts or food supplements containing flavonoids, significant levels of the phytochemicals can be achieved, similar to the concentrations effective in vitro (Aszalos 2008).

Devil's claw has been reported by Unger and Frank (2004) to be an inhibitor of some CYP isoenzymes, suggesting that possible interactions might arise with conventional drugs metabolized by these CYPs. Since many of these drugs are transported by P-gp, the present study suggests that also the ability of Devil's Claw to modulateactivity/expression of P-gp should be taken into account.

Therefore, not only further studies are required in order to identify the active components as well as to understand the molecular mechanisms underlying P-gp modulation by Devil's Claw, but also the potential for clinical implications of Devil's Claw- P-gp interaction needs a careful consideration.

Acknowledgements

This work was supported by University of Pisa, Italy (Fondi di Ateneo 2007).

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Nadia Romiti (a), Gianfranco Tramonti (b), Alessandro Corti (a), Elisabetta Chieli (a), *

(a) Dipartimento di Patologia Sperimentale, Sezione di Patologia Generate e Clinica. Universita degli Studi di Pisa, Pisa, Italy

(b) Dipartimento di Medicina Interna, Sezione di Nefrologia, Universita degli Studi di Pisa, Pisa, Italy

* Corresponding author. Tel.: +390502218558; fax: +390502218557.

E-mail address: chieli@biomed.unipi.it (E. Chieli).

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Author:Romiti, Nadia; Tramonti, Gianfranco; Corti, Alessandro; Chieli, Elisabetta
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
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Geographic Code:4EUIT
Date:Dec 1, 2009
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