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Transport of sennosides and sennidines from Cassia angustifolia and cassia senna across Caco-2 monolayers--an in vitro model for intestinal absorption.

Abstract

Laxative effects of Senna preparations are mainly mediated by rheinanthrone, a metabolite formed in the intestinal flora from dianthrones. Nevertheless, it was not clear whether dianthrones are bioavailable at all and contribute to the overall effects of this important medicinal plant. Using the Caco-2 human colonic cell line as an in vitro model of the human intestinal mucosal barrier, the bioavailability of dianthrones was studied in apical to basolateral (absorptive) and basolateral to apical (secretive) direction. Permeability coefficients ([P.sub.c]) and percent transport were calculated based on quantitations by HPLC. From the data obtained it was concluded that sennosides A and B, as well as their aglycones sennidine A and B are transported through the Caco-2 monolayers in a concentration-dependent manner and their transport was linear with time. The absorption in apical to basolateral direction was poor and [P.sub.c] values were comparable to mannitol. The transport was higher in the secretory direction, indicating a significant efflux (e.g. by efflux pumps) of the (poorly) absorbed compounds in the intestinal lumen again. Our findings support the general understanding that the laxative effects of Senna are explainable mainly by metabolites and not by the natively present dianthrones.

[c]2007 Elsevier GmbH. All rights reserved.

Keywords: Cassia spp; Senna; Sennosides; Caco-2; Intestinal bioavailability

Introduction

Cassia angustifolia VAHL. (Fabaceae) and C. senna L. are two morphologically closely related species, native to Northern Africa and Arabia (Wichtl, 2002). They differ in size (C. angustifolia is taller), but both show pinnate leaves and yellow flowers, and also contain the same laxative constituents. In Senna fruits and leaves hydroxyanthracene derivatives (dianthrones) are found, which are present as glycosides (sennosides) mainly; respective aglycones are called sennidines. Dianthrones are not natively present compounds but formed enzymatically in the plant during drying at low temperatures (Wagner, 1999). They act as prodrugs, and pass through the upper part of the gastrointestinal tract nearly unaltered to reach the caecum and colon, where they are transformed to rheineanthrone, the active metabolite. Mode of action via rheinanthrone formation is well accepted and understood. The systemic availability of this compound is less than 5%; yet, it potentially activates peristaltis of the colon, resulting in laxative effects (Krumbiegel and Schulz, 1993; Raimondi et al., 2002).

In a recent publication by Laitinen et al., it has been shown that anthranoid laxatives influence the absorption of poorly permeable drugs in vitro (Laitinen et al., 2007). What remains unclear is the question whether the natively present dianthrones (most important are sennosides A and B, and their aglyca; Fig. 1) are absorbed to a certain extent on their way to the colon (which takes approx. 8-10 h) or not. Especially, the less polar sennidines seem to be suited for absorption and therefore might play a significant role in the overall pharmacological effects of Senna.

[FIGURE 1 OMITTED]

Caco-2 cell monolayers are a well-accepted in vitro model for the evaluation of drugs for their intestinal absorption potential (Yee, 1997). Under normal conditions, Caco-2 cells spontaneously differentiate to mature cells which form intact monolayers (Artursson and Karlsson, 1991). The adjacent cells adhere through tight junctions formed at the apical side of the monolayer, which can discriminate the transcellular and paracellular transport of drugs across the epithelial layer (Yamashita et al., 2000). Although Caco-2 cells originate from human colon carcinoma, they acquire many features of absorptive intestinal cells during culture such as microvillus structure, metabolic enzymes and carrier-mediated transport systems for sugars, amino acids and several drugs. They show morphological and biochemical similarity to normal intestinal enterocytes, so that drug permeability through Caco-2 monolayers usually correlates well with the intestinal membrane in vivo (Khan et al., 2003). Yet, limitations of this in vitro assay are low levels of cytochrome P450 3A4, lack of a mucus layer, variable expression of transportes and nonspecific drug binding (Shah et al., 2006; Khan et al., 2004; Lim and Lim, 2006).

In this study, the Caco-2 model was utilized to investigate the intestinal permeability of major Senna dianthrones.

Materials and methods

Materials

Sennosides A and B and sennidine A were purchased from Chromadex, Santa Ana, CA, USA, and sennidine B was from Indofine, Belle Mead, NJ, USA. Atenolol, Krebs-Ringer bicarbonate buffer, D-(+)-glucose and DMSO were from Sigma (St. Louis, MO, USA). Caco-2 cells were obtained from the American Tissue Culture Collection (ATCC), Manassas, VA, USA, cell culture media and reagents were purchased from Gibco (Grand Island, NJ, USA). All other chemicals (p.a. grade) and solvents (analytical grade) were obtained from Fisher Scientific (Fair Lawn, NJ, USA). Transwell[TM] plates were purchased from Corning (Corning, NY, USA).

Cell culture and Caco-2 monolayers

Caco-2 cells were grown in modified Eagle's minimum essential medium (MEM), supplemented with 20% bovine calf serum (BCS) and antibiotics (penicillin and streptomycin) as described earlier (Khan et al., 2003). Upon confluency the cells were harvested with trypsin-EDTA, and seeded onto the Transwell inserts (4.7 [cm.sup.2] surfaces, pore size of 0.4 [micro]m) at a density of 5 x [10.sup.4] cells/ insert. After 21-23 days in culture the monolayers were used for the transport experiments. Their integrity was determined by examining the cells using a light microscope and by measuring trans-epithelial electrical resistance (TEER) across the monolayers with a Millicell-ERS system (Millipore, Billerica, MA, USA).

Transport experiments

Only monolayers displaying a TEER value equal or above 300 [OMEGA] x [cm.sup.2] were utilized for the transport experiments. Krebs-Ringer bicarbonate buffer (pH 7.4) containing 40 mM glucose was used as transport medium. A 10 mM stock solution of the test compound was prepared in DMSO and diluted with transport medium to the desired concentrations (100 and 200 [micro]M) prior to the experiment.

The experiment was initiated by replacing the transport medium on the donor side (apical side, for apical to basolateral transport; basolateral side, for basolateral to apical transport) with medium containing the test compound. Thereafter, 200 [micro]l portions of samples were taken out from the receiver side every hour for 5 h. The volume was kept constant by adding fresh transport medium to the receiver side. At the end of the experiment, 200[micro]l were also taken out from the donor side. To account for a possible uptake of the test compounds by Caco-2 cells the medium was removed from both sides, and the monolayer was washed with transport medium. Then, 1 ml of methanol was added to both sides, and the absorbed compound was extracted by shaking the plate for 30 min (Walgren et al., 1998).

Analytical method

Atenolol was analyzed by an HPLC method as described earlier (Chong et al., 1997). For the analysis of dianthrones a new method was developed in-house, utilizing a Luna C18(2) column (150 x 4.6 mm, 5[micro]m) from Phenomenex (Torrance, CA, USA). The mobile phase comprised water (A) and acetonitrile (B), both containing 0.5% acetic acid. Gradient elution was performed by changing the composition from 9A/1B to 1A/9B within 20 min, at a flow rate of 1 ml/min, 30 [degrees]C column temperature and a detection wavelength of 360 nm, respectively.

For generating calibration curves, a stock solution of the test compounds (1 mg/ml each) was prepared in acetone: water (7:3) and serially diluted with methanol. Within a concentration range of 0.1-100 [micro]g/ml the HPLC method was linear ([R.sup.2][greater than or equal to]0.998), sensitive (LOD[less than or equal to] 0.05[micro]g/ml) and accurate (recovery rates [greater than or equal to]97.9%; data not shown in detail) for all compounds investigated.

Transport experiments were performed in triplicates, respective results are presented in Table 1 as mean [+ or -] SD. Statistical significance of the results was evaluated using the Students t-test(p<0.05).
Table 1. Transport parameters of sennosides and sennidines in the
Caco-2 assay ([p.sub.c] = permeability coefficient, % T =
percent transport, Rec = recovery; data are presented as mean [+ or -]
SD, n = 3)

 Apical to basolateral (absorptive)

Compound Cone. [P.sub.c] x %T Rec (%)
 [10.sup.-6]
 cm/sec

Sennoside 100 3.09[+ or -] 1.20[+ or -] 101.76[+ or -]
A [micro]M 0.23 0.06 0.52

 200 4.77[+ or -] 1.84[+ or -] 94.10[+ or -]
 [micro]M 0.04 0.01 2.19

Sennoside 100 2.57[+ or -] 0.96[+ or -] 104.51[+ or -]
B [micro]M 0.42 0.13 3.92

 200 5.21[+ or -] 1.89[+ or -] 93.91[+ or -]
 [micro]M 1.07 0.30 0.56

Sennidine 100 5.85[+ or -] 2.11[+ or -] 81.70[+ or -]
A [micro]M 0.06 0.01 3.58

 200 9.09[+ or -] 3.25[+ or -] 89.85[+ or -]
 [micro]M 0.57 0.20 3.29

Sennidine 100 5.01[+ or -]0.11 1.87[+ or -] 92.28[+ or -]
B [micro]M 0.07 0.55

 200 5.80[+ or -] 2.14[+ or -] 90.01[+ or -]
 [micro]M 0.99 0.32 2.24

atenolol (a) 200 13.7[+ or -] 4.95[+ or -] n.d.
 [micro]M 0.68 2.59

 Basolateral to apical (secretive)

Compound [P.sub.c] x %T Rec (%)
 [10.sup.-1]
 cm/sec

Sennoside 2.23[+ or -] 0.30 0.92[+ or -] 93.52[+ or -]
A 0.07 1.93

 8.10[+ or -] 0.64 3.23[+ or -] 0.4 91.36[+ or -]
 4.09

Sennoside 7.45[+ or -] 0.26 2.86[+ or -] 99.43[+ or -]
B 0.06 8.35

 14.37[+ or -]1.66 5.51[+ or -]0.28 96.75[+ or -]
 1.38

Sennidine 6.29[+ or -] 0.75 2.42[+ or -] 87.86[+ or -]
A 0.15 0.06

 17.64[+ or -]1.82 6.55[+ or -] 95.26[+ or -]
 0.12 1.85

Sennidine 8.47[+ or -] 0.64 3.08[+ or -] 91.36[+ or -]
B 0.19 2.12

 16.20[+ or -] 5.93[+ or -] 97.65[+ or -]
 0.78 0.32 1.30

atenolol 16.6 [+ or -] 8.15[+ or -] n.d.
(a) 0.60 2.62

(a)Model compound for passive transport.


Results and discussion

Because of the widespread use of Senna preparations, this study was undertaken to investigate the intestinal transport and uptake of dianthrones, the laxatives natively present in this plant. Using Caco-2 cell monolayers as a model for the intestinal mucosa, permeability coefficients ([P.sup.c[) and percent transport (%T) were determined for sennosides A and B as well as sennidines A and B, at concentrations of 100 and 200 [micro]M. The transport was monitored in absorptive (apical to basolateral) and secretory (basolateral to apical) direction. Atenolol, a model compound for passive diffusion was included in every experiment in duplicate. When the permeability coefficient and transport rate for this compound were below 4.0 x [10.sup.-6]cm/s and 15%, the cell monolayers were considered tight (Shah et al., 2006).

Permeability in both directions was calculated by linear regression analysis of the cumulative amount transported based on the equation formulated previously (Walle et al., 1999) [P.sup.c]=dQ/dtX1/60X1/AX1/C ([P.sub.c] = permeability coefficient in cm/s, dQ/dt=rate of appearance of the drug on the receiver side in [micro]g/min, [C.sub.o] = initial drug concentration on the donor side in [micro]g/ ml, A=surface area of the monolayer). Percent transport (%T) was calculated as the ratio of cumulative concentration in the receiver to the donor side x 100. Transport was monitored for a period of 5 h. By plotting the cumulative amounts versus time a linear correlation was observed for all tested compounds in absorptive as well as secretive direction (Fig. 2 shows the transport characteristics of sennoside A and sennidine A as examples).

[FIGURE 2 OMITTED]

The transport parameters ([P.sub.c] and %T) for sennosides and sennidines, determined at concentrations of 100 and 200 [micro]M, are summarized in Table 1. [P.sub.c] values for sennoside A and B ranged from 2.6 X [10.sup.-7] to 5.2 x [10.sup.-7] cm/s in apical to basolateral direction, and 2.2 x 10.sup.-7] to 14.4 X [10.sup.-7] in basolateral to apical direction, respectively. For sennidine A and B, [p.sup.c] values varied from 5.0 X [10.sup.-7] to 9.1 X [10.sup.-7] in apical to basolateral, and 6.3 X [10.sup.-7] in basolateral to apical direction. The overall permeability of sennidines A and B was higher than the corresponding glycosides in both directions. As seen in Table 1, permeability coefficients and % transport were concentration dependent. In comparison to atenolol ([p.sub.c] value 13.7 X [10.sup.-7] cm/s) permeability of the tested compounds was much lower in apical to basolateral direction, indicating a poor absorption through intestinal mucosa. However, the [p.sub.c] values are comparable to the reported values for mannitol (5 X [10.sup.-7] cm/s), which is a model compound for poor absorption and is known to be exclusively absorbed through the paracellular route (Chong et al., 1997). Propanolol, a transcellular marker with lipophilic character, shows a [p.sub.c] value of 230 X [10.sup.-7] cm/s, while glucose follows an active transport mechanism. Its [p.sub.c] value is reported to be 368 X [10.sup.-7] cm/s in the apical to basolateral direction (Walgren et al., 1998).

When transport rates were monitored in the secretory (basolateral to apical) direction, comparatively higher permeability coefficients and % transport were observed (except for sennoside A at 100 [micro]M; Table 1). These results indicated a trend of lower transport in the absorptive direction than in the secretory direction, leading to an efflux of the drugs. Further studies will be needed to fully understand the mode of intestinal transport of these compounds, but the involvement of efflux pumps can be considered.

The transport properties of aglycones were better than their corresponding glycosides, as aglycones generally showed a higher permeability coefficient and rate of transport in the absorptive as well as in the secretory direction (Table 1).

At the end of the experiments (after 5 h), the cells were extracted with methanol and analyzed for possible remains of the dianthrones. No significant degradation, metabolism or binding of the test compounds to Caco-2 cells was observed, as the recovery rates were higher than 90% in most cases with the exception of sennidine A (100[mue]M), where less than 90% could be recovered.

Results of this study indicate poor transport of sennosides A and B and their aglycones across Caco-2 monolayers, with aglycones generally showing a higher permeability than glycosides. Additionally, higher transport rates were found in the secretory direction compared to the absorptive one. This indicates the involvement of efflux pumps, and further diminishes a possible absorption of dianthrones in the upper gastrointestinal tract. These in vitro observations are in agreement with the hypothesis that natively presents sennosides and sennidines are resorbed in the intestine to a very small extent only, so that they will not contribute to the laxative effects mediated by metabolites (rheineanthrone) significantly. Our results therefore confirm the commonly practiced mode of application and dosage of Senna preparations.

Acknowledgment

The authors would like to thank Ms. Shatara Porchia-Whilte for her technical assistance.

References

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* Corresponding author. Tel.: +43 512 507 5307; fax: +435125072939.

E-mail address: Markus.Ganzera@uibk.ac.at (M. Ganzera).

0944-7113/$--see front matter [c] 2007 Elsevier GmbH. All rights reserved. doi: 10.1016/j.phymed.2007.03.008

B. Waltenberger (a), M. Ganzera (a),*, I.A. Khan (b), H. Stuppner (a), S.I. Khan (b)

(a) Department of Pharmacognosy, Institute of Pharmacy, University of Innsbruck, 6020 Innsbruck, Austria

(b) National Center for Natural Products Research, The University of Mississippi, University, MS 38677, USA
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Author:Waltenberger, B.; Ganzera, M.; Khan, I.A.; Stuppner, H.; Khan, S.I.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:1USA
Date:May 1, 2008
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