In Vitro Studies Indicate that Miquelianin (Quercetin 3-O-ß-D-Glucuronopyranoside) is Able to Reach the CNS from the Small Intestine

• Abstract
• Introduction
• Materials and Methods
• Chemicals
• Caco-2 cell culture
• Primary cultures of porcine brain capillary endothelial cells (PBCEC)
• Porcine chorioid plexus cell culture
• Uptake experiments with Caco-2 cells
• Permeability experiments
• Sample analysis in permeability experiments
• Results
• Discussion
• Acknowledgements
• References

Abstract

Miquelianin (quercetin 3-O-ß-D-glucuronopyranoside) is one of the flavonoids of St. John’s wort (Hypericum perforatum L.) whose antidepressant activity has been shown by the forced swimming test, an in vivo pharmacological model with rats. However, nothing is known about its ability to reach the CNS after oral administration. We examined the pathway of miquelianin from the small intestine to the central nervous system using three in vitro membrane barrier cell systems. In the Caco-2 cell line, miquelianin showed a higher uptake (1.93 ± 0.9 pmol × min^-1 × cm^-2) than hyperoside (quercetin 3-O-ß-D-galactopyranoside; 0.55 ± 0.18 pmol × min^-1 × cm^-2) and quercitrin (quercetin 3-O-α-L-rhamnopyranoside; 0.22 ± 0.08 pmol × min^-1 × cm^-2). The permeability coefficient of miquelianin (P _c = 0.4 ± 0.19 × 10 ^{- 6} cm/sec) was in the range of orally available drugs assuming sufficient absorption from the small intestine. Uptake and permeability of the examined compounds was increased by the MRP-2 inhibitor MK-571 indicating a backwards transport by this membrane protein. Porcine cell cultures of brain capillary endothelial cells were used as a model of the blood-brain barrier (bbb) and epithelial cells of the plexus chorioidei as a model of the blood-CSF barrier (bcb). Results indicate no active transport in one direction. Although moderate, the permeability coefficients (bbb: P _c = 1.34 ± 0.05 × 10 ^{- 6} cm/sec; bcb: P _c = 2.0 ± 0.33 × 10 ^{- 6} cm/sec) indicate the ability of miquelianin to cross both barriers to finally reach the CNS.

Introduction

Extracts of the upper parts of St. John’s wort (Hypericum perforatum L., Clusiaceae) are successfully used against mild and moderate depression as shown by many clinical studies [1]. Nevertheless, the active constituents of the extracts are not fully known. Besides phloroglucinols and naphthodianthrones, flavonoids seem to be important for the antidepressant activity of Hypericum perforatum [2]; not only was a flavonoid enriched fraction active in the Porsolt’s forced swimming test (FST), a validated animal model for the study of antidepressant drugs [3], but also some pure quercetin glycosides detected by bioguided fractionation showed significant activity in the FST [4]. The 3-O-ß-D-glucopyranoside (isoquercitrin), the 3-O-ß-D-galactopyranoside (hyperoside) and the 3-O-ß-D-glucuronopyranoside (miquelianin, Fig. [4]) of the aglycone quercetin were active in the FST whereas the aglycone itself and its 3-O-α-L-rhamnopyranoside (quercitrin) were not [4].

These results led to the hypothesis that, depending on the sugar side chain, differences exist in the absorption or the metabolism of these compounds. Results from in vivo and in vitro studies on quercetin glycosides indicate different metabolic routes; quercetin glucuronides including the 3-O-glucuronide were often detected as metabolic products [5], [6]. Because miquelianin was not only shown to be a native constituent of St. John’s wort but also a human metabolite after ingestion of various quercetin glycosides, the present study was performed in vitro to examine its ability to cross the membrane barriers from the small intestine to the CNS.

The human colonic cell line Caco-2 was used as an in vitro model of human intestinal absorption [7]. Porcine cell cultures of brain capillary endothelial cells were used as the blood-brain barrier model (bbb) [8]; epithelial cells of the plexus chorioidei were used as the blood-cerebrospinal fluid barrier (bcb) [9], [22].

Materials and Methods

Chemicals

Hyperoside and quercitrin were purchased from Roth KG, Karlsruhe (Germany); miquelianin was isolated as described previously [10]; all compounds were HPLC-pure. MK-571 was from Biomol Feinchemikalien GmbH, Hamburg (Germany) and D,L-propranolol from Sigma Aldrich Chemie, Steinheim (Germany).

Caco-2 cell culture

Cells were a gift from the Klinik und Polyklinik für Chirurgie, Westf. Wilhelms-University Muenster. They were grown in a humidified atmosphere with 5 % CO₂ at 37 °C and cultured in Earle’s medium 199 [PAA Lab., Linz (Austria)] with 10 % fetal bovine serum and 0.5 % of a penicillin (10 000 IU/mL)/streptomycin (10 000 μg/mL) mixture (Gibco BRL, Paisley, Scotland). The medium was exchanged three times a week. For all experiments cells at passages 10 to 25 were used.

Cells were seeded directly onto 12-well Transwell® inserts (1 cm², 0.4 μm pore size, 0.5 mL apical and 1.5 mL basolateral chambers, Transwell Costar, Badhoevedorp, Netherlands) and the culture medium was carefully replaced three times a week. 22 days post seeding, the integrity of the Caco-2 monolayers was tested by measuring their transepithelial electrical resistance (TEER) with an EVOM®-G-voltohmmeter (STX2-electrodes, World Precision Instruments, Berlin, Germany). Only monolayers with a TEER value > 250 Ω × cm² were used for further experiments. In earlier experiments a TEER-value of 250 Ω × cm² was correlated to a permeability coefficient (P _c) = 0.14 × 10 ^{- 6} cm/sec of FITC-dextran 4400 (0.1 mM), a commonly used control substance for low permeability and integrity of the monolayer [11]. As a control substance for high permeability propranolol × HCl (0.1 mM) showed a P _c = 37.5 ± 4.8 × 10 ^{- 6} cm/sec.

Primary cultures of porcine brain capillary endothelial cells (PBCEC)

Porcine brain capillary endothelial cells (PBCEC) were isolated and cultured according to [8]. Briefly, cerebra of freshly slaughtered pigs were mechanically homogenized and gradually digested by two proteases, together with further purification steps by density centrifugation. Cells were seeded onto collagen G-coated culture flasks and subcultivated on the third day in vitro (DIV 3) by fractionated trypsinization to reduce the number of contaminating pericytes.

Suspended PBCEC were seeded at a density of 200,000 cells/cm² on rat-tail collagen-coated polycarbonate filter inserts (Transwell No. 3401; 0.4 μm pore size, 1 cm² growth area; see above). The monolayers were cultivated as described by Franke et al. [8]. On the third day after passage complete medium was replaced by serum-free medium supplemented with 550 nM hydrocortisone. Medium exchange was performed extremely carefully to avoid damage of the monolayer. Like Caco-2 cells, PBCEC grow in a polarized way on the filter membranes [12], because the collagen coating of the filter mimics the basal membrane which surrounds the capillaries in vivo. Thus, the apical (donor) compartment of the filter insert represents the capillary lumen, whereas the basolateral (acceptor) chamber corresponds to the brain interstitial fluid.

In contrast to the Caco-2 TEER measurement, the transendothelial resistance of every PBCEC monolayer was quantified by impedance analysis [13] prior to permeation experiments. Only PBCEC monolayers showing a TEER > 900 Ω × cm² were used for permeability studies. In control experiments, TEER values above approx. 600 Ω × cm² correlated with a P _c for sucrose sufficiently low to perform reliable permeation assays (approx. 0.5 × 10 ^{- 6} cm/sec); besides FITC-dextran, sucrose is another commonly used low-molecular weight paracellular marker substance. For propranolol as a marker with good permeability, 100-fold higher Pc values were obtained (approx. 61.0 × 10 ^{- 6} cm/sec) [21].

Porcine chorioid plexus cell culture

Epithelial cells from porcine chorioid plexus were prepared as described [9]. Chorioid plexus tissue was taken from freshly slaughtered 6-month-old pigs and incubated with 0.25 % (w/v) trypsin solution (Biochrom, Berlin, Germany) for 2.5 h at 4 °C and then for 0.5 h at 37 °C. Unreleased tissue was separated and possibly liberated proteolytic activity was blocked by addition of newborn calf serum (Biochrom). Cells were spun down at 15 × g. The cells were resuspended in DMEM/HAM’s F12 (1 : 1) medium supplemented with 10 % FCS (PAA, Austria), 4 mM L-glutamine, 5 μg/mL insulin (Sigma), 100 μg/mL penicillin/streptomycin, then seeded on laminin (50 μg/mL in water, Sigma) coated filters (Transwell No. 3401; 0.4 μm pore size, 1 cm² growth area; see above). To suppress the growth of contaminating mainly fibroblastic cells, 20 μM cytosine arabinoside was added. The seeding density was adjusted such that 1 g of original tissue (wet weight) was used to inoculate 50 cm². The medium was changed every two days. After 9 days, the cells were cultured in serum-free medium for 4 to 5 days. Again medium was changed every second day. Like Caco-2 and PBCEC cells, the chorioid plexus cells grow in a polarised monolayer. But in contrast to the two models above, the apical chamber corresponds to the CSF side in vivo (acceptor), the basolateral to the blood side (donor). The transepithelial resistance of each filter was quantified by impedance analysis [13]. Only filter systems showing a TEER > 600 Ω × cm² were used for permeability measurements. In control experiments, this TEER value was correlated to a P_c = 0.2 × 10 ^{- 6} cm/sec for sucrose and a P_c = 42 × 10 ^{- 6} cm/sec for diazepam [22].

Uptake experiments with Caco-2 cells

The medium was exchanged with HBSS buffer pH 7.4 (Hanks’ balanced salt solution, 37 °C, H-8264, Sigma-Aldrich, UK) 30 min prior to analysis. For uptake studies, the buffer in the donor (apical) compartment was exchanged with prewarmed 100 μM solutions of miquelianin, hyperoside and quercitrin in HBSS. To test whether these flavonoids are targets for the MRP-2 transporter, 50 μM of the MRP-2 inhibitor MK-571 were added to the buffer and the flavonoid solutions. After 4 min, the uptake was stopped by removing the solutions and the cells were washed three times with 4 °C cold buffer. The flavonoids were extracted from the cells with methanol, 5 μL of an internal standard solution (pinocembrin 7-O-methyl ether in MeOH) were added and the volume was filled up to 2 mL with MeOH. Quantitative analysis was performed using a validated HPLC method as described [10].

Permeability experiments

For permeability experiments with the in vitro models 4 to 6 cell monolayers and 2 blank filters were used. The medium was exchanged with 37 °C warm buffer 30 min prior to analysis. Caco-2 experiments were carried out in HBSS buffer; CNS barrier experiments in Krebs-Ringer buffer pH 7.4 (KR) (1.1 mM magnesium chloride; 1.25 mM calcium chloride; 114 mM sodium chloride; 5 mM potassium chloride; 20 mM sodium bicarbonate; 10 mM HEPES and 25 mM glucose). The buffer of the donor compartment (apical chamber for Caco-2 and bbb; basolateral chamber for bcb) was then exchanged with a prewarmed 100 μM solution of miquelianin or 100 μM miquelianin + 50 μM MK-571, respectively. After 15, 30, 45 and 60 min (Caco-2 experiments also after 90 min) 200 μL samples were taken from the acceptor compartment and the missing volume supplemented with prewarmed buffer to maintain sink conditions. The samples were analyzed by HPLC.

The permeability coefficient P was calculated with the equation:[]

where d_c/d_t represents the change of concentration in the acceptor compartment, V_u the volume of the acceptor chamber, A the membrane surface area and c₀ the initial concentration in the donor chamber [14]. To consider the effect of the polycarbonate membrane the permeability coefficients of miquelianin obtained with the blank filters were used to correct the P values of miquelianin with the cell monolayers:[]

where P _c is the P value of the monolayer, P _f the P value of the blank filter (Caco-2 : 8.3 × 10 ^{- 5} cm/sec; bbb: 4.2 × 10 ^{- 5} cm/sec; bcb: 1.1 × 10 ^{- 4} cm/sec) and P _{c + f} represents the investigated P value of miquelianin with filter and monolayer [14].

For transport studies at the CNS barriers (n = 6), the apical and basolateral buffer solutions were exchanged by 100 μM miquelianin in buffer. 100 μL samples of both chambers were collected after 30 min, 5 h and 24 h and quantified by HPLC. Active transport and uptake into the cells and the filter were calculated from the change of concentration in both chambers with and without monolayers (2 blank filters).

Sample analysis in permeability experiments

All samples of the permeability experiments were quantified by HPLC optimized for miquelianin with a Waters 2690 Alliance™ separations module, a Waters 996 PDA detector (detection wavelength: 284 nm), a Supelco Discovery™ C-18 column (25 × 4.6 mm; 5 μm) and a linear gradient from 10 % B to 60 % B in 15 min (A: Aqua millipore®/tetrahydrofuran/trifluoroacetic acid (97 + 2 + 1); B: acetonitrile/tetrahydrofuran/trifluoroacetic acid (97 + 2 + 1); flow: 1 mL/min; injection volume: 100 μL). Data analysis was carried out with Millenium³² Version 3.20 (Waters Corporation). To quantify miquelianin a calibration curve was performed with different dilutions of miquelianin in KR buffer (3.2 ng/mL to 49.6 μg/mL). With the resulting equation (y = 77.978x - 2617.1; r² = 0.999) the concentration of miquelianin is calculated ( = x [ng/mL]; t_r = 8.4 min; detection limit 1 ng/mL).

Results

Fig. [1] shows the apparent uptake rates of the flavonols at 100 μM into the Caco-2 cells after 4 min incubation time with 0.22 ± 0.08 pmol × min^-1 × cm^-2 for quercitrin, 0.55 ± 0.18 pmol × min^-1 × cm^-2 for hyperoside and 1.93 ± 0.9 pmol × min^-1 × cm^-2 for miquelianin. Uptake rates increased in the presence of 50 μM of the MRP-2 inhibitor MK-571 to 3.0 ± 1.89 pmol × min^-1 × cm^-2 for quercitrin, 23.1 ± 9.97 pmol × min^-1 × cm^-2 for hyperoside and 11.2 ± 0.96 pmol × min^-1 × cm^-2 for miquelianin (mean ± SE; n = 3 or 4).

Miquelianin (Fig. [4]) was used for permeability experiments in all three barrier cell systems (Fig. [2]). The permeability coefficients (P _c) obtained for miquelianin with Caco-2 cells was 0.4 ± 0.19 × 10 ^{- 6} cm/sec. In the presence of 50 μM MK-571 the P _c value increased up to 0.9 ± 0.22 × 10 ^{- 6} cm/sec. With the PBCEC monolayers which represent the blood-brain barrier a P _c-value of 1.34 ± 0.05 × 10 ^{- 6} cm/sec was observed for miquelianin. Porcine chorioid plexus epithelial cells which mimick the blood-cerebrospinal fluid barrier gave a P _c value of 2.0 ± 0.33 × 10 ^{- 6} cm/sec for the same flavonoid (mean ± SEM; n = 4 to 6).

With the two CNS barriers no active transport in one direction could be detected by measuring the distribution of the apical and basolateral offered miquelianin over 24 h (Fig. [3]).

Discussion

Walgren et al. showed with the Caco-2 cell model that quercetin 4′-O-ß-glucoside is a substrate for the intestinal sodium-dependent D-glucose cotransporter (SGLT1) [15] but also a substrate for the multidrug resistance associated protein-2 (MRP-2) [16]. This suggests that other flavonol glycosides may also be transported into these cells via the SGLT1 and backwards by the MRP-2. Using the same in vitro model, we examined the uptake of three quercetin glycosides present in St. John’s wort extracts into Caco-2 cells in order to measure possible differences in the amount of their uptake: miquelianin, the 3-O-ß-D-glucuronopyranoside was the compound with the highest uptake, followed by the 3-O-ß-D-galactopyranoside (hyperoside) and the 3-O-α-L-rhamnopyranoside (quercitrin), the latter of which was not significantly active in the FST for antipressant activity [4]. The uptake of miquelianin was about four-fold higher than that of hyperoside which itself was almost two-fold higher than that of quercitrin (Fig. [1]). In view of the low transport of the α-L-rhamnoside, the data not only support the preference of the SGLT1 for ß-D-hexosides but also their active transport, as by passive transport the more lipophilic rhamnoside would have had significantly higher values than the two hexosides, in particular the glucuronide. The uptake values of the three quercetin glycosides increased in the presence of the MRP-2 inhibitor MK-571 (Fig. [1]). With its 6-fold increase miquelianin is a poor substrate when compared to hyperoside with a 40-fold increase; it is obvious that the relatively good uptake of miquelianin without MK-571 is caused by its comparatively poor backwards transport.

Miquelianin (Fig. [4]), a pharmacologically active substance of St. John’s wort and a metabolite of some quercetin glycosides in humans, was chosen for further permeability experiments to investigate its behaviour at cellular barriers. With the Caco-2 cell line, that has a good in vitro/in vivo-correlation [7], [17], its permeability coefficient P _c [cm/sec] in the small intestine was investigated. The relatively polar compound miquelianin at 100 μM showed a P _c value of 0.4 ± 0.19 × 10 ^{- 6} cm/sec. Artursson and Karlsson correlated P _c values of 0.1 to 1 × 10 ^{- 6} cm/sec with a drug absorption in vivo of 1 to 100 % [7]; Yee predicted a drug absorption of 20 % for the same P _c value range [17]. The permeability of miquelianin, though moderate, is in the range of commonly used drugs such as terbutaline (P _c = 0.5 × 10 ^{- 6} cm/sec) or ranitidine (P _c = 0.5 × 10 ^{- 6} cm/sec) [18] and indicates that miquelianin can be absorbed from the small intestine. For quercetin 4′-O-glucoside and quercetin 3,4′-O-diglucoside poor permeability coefficients of P _c < 0.02 × 10 ^{- 6} cm/sec and 0.09 ± 0.03 × 10 ^{- 6} cm/sec were obtained from the apical to the basolateral transport [19]. These data again support the influence of the type of sugars and their substitution position on the absorption of flavonoid glycosides. As in the uptake experiments, the P _c value of miquelianin expectedly increased in the presence of the MRP-2 inhibitor MK-571 to P _c = 0.9 ± 0.22 × 10 ^{- 6} cm/sec (Fig. [2]).

An antidepressant compound should reach the CNS. Thus, two CNS barriers were chosen for an in vitro investigation: a primary culture of porcine brain capillary endothelial cells (PBCEC) for the blood-brain barrier (bbb) and a porcine chorioid plexus epithelial cell culture for the blood-cerebrospinal fluid barrier (bcb); both monolayers represent the barrier system for substances to reach the CNS [20]. Using these cell systems, similar P _c values were obtained for miquelianin with the Caco-2 cell system: 1.34 ± 0.05 × 10 ^{- 6} cm/sec for the bbb and 2.0 ± 0.33 × 10 ^{- 6} cm/sec for the bcb (Fig. [2]). Its permeability across the bbb is in the range of the CNS active alkaloid morphine with P _c = 1.6 ± 0.3 × 10 ^{- 6} cm/sec [21]. Although moderate, the permeability coefficients indicate that miquelianin is able to cross both barrier systems to exert pharmacodynamic effects in the CNS. However, active transport processes could not be detected (Fig. [3]), as no significant shift of miquelianin could be observed between the different compartments when the compound was added to the apical and basolateral chamber simultaneously.

In summary, we have shown in vitro, that the in vivo antidepressant flavonol glucuronopyranoside miquelianin of St. John’s wort is able to cross the important barriers from the small intestine to the CNS. The data are also valid for orally administered quercetin glycosides of other herbal medicinal products or functional food. Differences in Caco-2 uptake of quercetin glycosides and so possible differences in absorption could be correlated with different kinds of glycosidic partners. The participation of MRP-2 in the absorption processes was shown.

Acknowledgements

Thanks are due to Dr. F. Petereit, Dr. M. Lechtenberg and to Dr. J. Wegener for helpful discussions.

References

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Dr. Guido Juergenliemk

Institute of Pharmaceutical Biology and Phytochemistry

Westf. Wilhelms-University

Hittorfstr. 56

49149 Muenster

Germany

Phone: +49-251-833-3376

Fax: +49-251-833-8341

Email: jurgenl@uni-muenster.de

References

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  PubMedSearch in Google Scholar
• 2 Nahrstedt A. Antidepressant constituents of Hypericum perforatum . In: Chrubasik S, Roufogalis BD, editors Herbal medicinal products for the treatment of pain. Lismore NSW; Southern Cross University Press 2000: pp. 144-53
  Search in Google Scholar
• 3 Butterweck V, Christoffel V, Nahrstedt A, Petereit F, Spengler B, Winterhoff H. Step by step removal of hyperforin and hypericin: Activity profile of different Hypericum preparations in behavioural models.  Life Sciences. 2003;  73 627-39
  PubMedSearch in Google Scholar
• 4 Butterweck V, Juergenliemk G, Nahrstedt A, Winterhoff H. Flavonoids from Hypericum perforatum show antidepressant activity in the forced swimming test.  Planta Med. 1999;  66 3-6
  Thieme ConnectPubMedSearch in Google Scholar
• 5 Gee J M, Dupont M S, Day A J, Plumb G W, Williamson G, Johnson I T. Intestinal transport of quercetin glycosides in rats involves both deglycosylation and interaction with the hexose transport pathway.  J Nutr. 2000;  130 2765-71
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• 6 Graefe E U, Wittig J, Mueller S, Riethling A K, Uehleke B, Drewelow B, Pforte H, Jacobasch G, Derendorf H, Veit M. Pharmacokinetics and bioavailability of quercetin glycosides in humans.  Herbal Med. 2001;  41 492-9
  CrossrefPubMedSearch in Google Scholar
• 7 Artursson P, Karlsson J. Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells.  Biochem Biophys Res Commun. 1991;  175 880-5
  CrossrefPubMedSearch in Google Scholar
• 8 Franke H, Galla H J, Beuckmann C T. Primary cultures of brain microvessel endothelial cells: a valid and flexible model to study drug transport through the blood-brain barrier in vitro .  Brain Res Prot. 2000;  5 248-56
  CrossrefPubMedSearch in Google Scholar
• 9 Gath U, Hakvoort A, Wegener J, Decker S, Galla H J. Porcine choroid plexus cells in culture: expression of polarized phenotype, maintenance of barrier properties and apical secretion of CSF-components.  Europ J Cell Biol. 1997;  74 68-78
  PubMedSearch in Google Scholar
• 10 Juergenliemk G, Nahrstedt A. Phenolic compounds from Hypericum perforatum L.  Planta Med. 2002;  68 88-91
  Thieme ConnectPubMedSearch in Google Scholar
• 11 Boje K. Phytochemische und biopharmazeutische Untersuchungen an Harpagophytum procumbens DC. PhD Thesis 2002 Münster, Germany;
  Search in Google Scholar
• 12 Tewes B, Galla H J. Lipid polarity in brain capillary endothelial cells.  Endothelium. 2001;  8 207-20
  PubMedSearch in Google Scholar
• 13 Wegener J, Hakvoort A, Galla H J. Barrier function of porcine choroid plexus epithelial cells is modulated by cAMP-dependent pathways in vitro .  Brain Res. 2000;  853 115-24
  CrossrefPubMedSearch in Google Scholar
• 14 Artursson P, Karlsson J, Ocklind G, Schipper N. Studying transport process in absortive epithelia. In: Shaw AJ Epithelia cell culture - a practical approach. RL Press 1996: pp. 111-33
  Search in Google Scholar
• 15 Walgren R A, Lin J T, Kinne R KH, Walle T. Cellular uptake of dietary flavonoid quercetin-4′-ß-glucoside by sodium-dependent glucose transporter SGLT1.  J Pharmacol Exp Ther. 2000;  294 837-43
  PubMedSearch in Google Scholar
• 16 Walgren R A, Karnaky K J, Lindenmayer G E, Walle T. Efflux of dietary flavonoid quercetin-4′-ß-glucoside across human intestinal Caco-2 cell monolayers by apical multidrug resistance-associated protein-2.  J Pharmacol Exp Ther. 2000;  294 830-6
  PubMedSearch in Google Scholar
• 17 Yee S. In vitro permeability across Caco-2 cells (colonic) can predict in vivo (small intestinal) absorption in man - fact or myth.  Pharm Res. 1997;  14 763-6
  CrossrefPubMedSearch in Google Scholar
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Dr. Guido Juergenliemk

Institute of Pharmaceutical Biology and Phytochemistry

Westf. Wilhelms-University

Hittorfstr. 56

49149 Muenster

Germany

Phone: +49-251-833-3376

Fax: +49-251-833-8341

Email: jurgenl@uni-muenster.de