Permeability and Conjugative Metabolism of Flaxseed Lignans by

Jan 13, 2014 - Flaxseed contains significant levels of the lignan, 1, which is known to undergo conversion to its aglycone, 2, and further biotransfor...
0 downloads 9 Views 866KB Size
Article pubs.acs.org/jnp

Permeability and Conjugative Metabolism of Flaxseed Lignans by Caco‑2 Human Intestinal Cells Jatinder Kaur Mukker,† Deborah Michel,† Alister D. Muir,‡ Ed S. Krol,† and Jane Alcorn*,† †

Drug Discovery and Development Research Group, College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5C9, Canada ‡ Agriculture and Agri-Food Canada, Saskatoon, Saskatchewan S7N 0X2, Canada ABSTRACT: Reports in the literature associate the dietary intake of flaxseed lignans with a number of health benefits. The major lignan found in flaxseed, secoisolariciresinol diglucoside (1), undergoes metabolism principally to secoisolariciresinol (2), enterodiol (3), and enterolactone (4) in the human gastrointestinal tract. Systemically, lignans are present largely as phase II enzyme conjugates. To improve understanding of the oral absorption characteristics, a systematic evaluation of the intestinal permeation was conducted and the conjugative metabolism potential of these lignans using the polarized Caco-2 cell system was analyzed. For permeation studies, lignans (100 μM) were added to acceptor or donor compartments and samples were taken at 2 h. For metabolism studies, lignans (100 μM) were incubated in Caco-2 for a maximum of 48 h. Cell lysates and media were treated with β-glucuronidase/sulfatase, and lignan concentrations were determined using HPLC. Apical-to-basal permeability coefficients for 2−4 were 8.0 ± 0.4, 7.7 ± 0.2, and 13.7 ± 0.2 (×10−6) cm/s, respectively, whereas efflux ratios were 0.8−1.2, consistent with passive diffusion. The permeation of compound 1 was not detected. The extent of conjugation after 48 h was 99% for 1−4, respectively. These data suggest 2−4, but not 1 undergo passive permeation and conjugative metabolism by Caco-2 cells.

P

colonic microflora to the mammalian lignans enterodiol (3) and enterolactone (4).20 Although attempts have been made in the literature to draw an epidemiological association between 4 and the positive health effects of flaxseed consumption,21 it still remains controversial whether plant-derived lignans (i.e., 1 and 2) and/or the mammalian lignans (i.e., 4 and 3) mediate the putative health benefits associated with flaxseed lignan consumption. Hence, knowledge of the biological fate of orally ingested lignans is a prerequisite to an understanding of their role in protection against or mitigation of chronic diseases. A limited number of studies have reported pharmacokinetic information on flaxseed lignans.22−25 Collectively, these studies indicate that following the oral administration of purified 1 or 2, 1 was not detected and very low blood levels of 2, 3, and 4 were observed and these existed primarily as phase II metabolites. Furthermore, 3 and 4 appeared systemically 8−10 h following the oral administration of 1, indicating the need for their conversion by bacteria of the colon.22 Interestingly, despite these low levels, oral administration of 4 to rats (10 mg/kg) for seven weeks inhibited mammary tumor growth in a tumor rat model.26 One study demonstrated that fecal excretion was the most significant elimination route for both the plant-derived and mammalian lignans.27 Low systemic levels and significant

lant polyphenolic compounds continue to receive prominent attention for their health benefits in a number of chronic disease states.1 Such attention has intensified research efforts to confirm efficacy and to elucidate the mechanisms underlying their bioactivity in chronic disease. Lignans, a class of phenylpropanoid compounds widely distributed in the plant kingdom, have generated particular interest due to their ability to favorably modulate biological risk factors of cardiovascular disease2 and cancer.3,4 Flaxseed (Linum usitatissimum L.; Linaceae) is a rich source of the lignan secoisolariciresinol diglucoside (1),5 and human clinical trials and animal model studies have reported important beneficial cardiovascular effects of flaxseed lignan supplementation such as improvement in blood lipid profiles,6,7 reduction in the development of aortic atherosclerosis,8−10 and delayed onset of type II diabetes.11−13 Furthermore, several studies suggest a protective role for flaxseed lignans against cancers of the breast,3,14,15 prostate,16,17 and colon.18 In the flaxseed, 1 exists as an oligomer with ester linkages to 3-hydroxy-3-methylglutaric acid, cinnamic acid, and other phenolic glucosides.19 It is assumed, but not yet demonstrated, that when the ester-linked 1 containing oligomer is ingested, the first metabolic step is the cleavage of the ester linkage and the release of free 1. Subsequently, the glucosidic groups are cleaved from 1 to yield the aglycone form, secoisolariciresinol (2), in the upper gastrointestinal tract. Compound 2 either is absorbed or will undergo subsequent metabolism by the © 2014 American Chemical Society and American Society of Pharmacognosy

Received: July 12, 2013 Published: January 13, 2014 29

dx.doi.org/10.1021/np4004905 | J. Nat. Prod. 2014, 77, 29−34

Journal of Natural Products

Article

absorption of compounds from the gastrointestinal tract lumen into the systemic blood depends upon the ability of compounds to permeate across the intestinal barrier and bypass presystemic metabolism in the intestine and liver. As an important step to an enhanced understanding of the oral absorption properties of 1 and its metabolites, 2−4, a systematic evaluation of lignan permeation across intestinal cells and the contribution of intestinal cells to phase II metabolism of 1 and its metabolites, 2−4, was conducted. This evaluation involved the use of the Caco-2 cell monolayer, as its use is widely accepted as an intestinal permeability screening assay31 and has been used to gain preliminary understandings on the propensity for intestinal phase II enzyme metabolism in an intact cellular system.42 Secoisolariciresinol Diglucoside (1) Does Not Permeate across the Caco-2 Polarized Epithelium. The bidirectional permeation of lignans across the Caco-2 monolayer was investigated to determine an apparent permeability coefficient and efflux ratio (Table 1). Since the HPLC analytical sensitivity

fecal excretion suggest poor oral bioavailability of lignans, which may be due to poor permeation characteristics, extensive presystemic metabolism, or poor release of 1 due to inadequate hydrolysis of the ester linkages.28,29 Since lignans are known to undergo phase II conjugative metabolism (glucuronidation and sulfation) and enterohepatic recirculation,28,29 with a very minor contribution by cytochrome P450-mediated oxidative metabolism pathways,24,30 knowledge of the permeation characteristics of lignans and their presystemic metabolism by phase II conjugative reactions will be critical to an understanding of the biologically active lignan form. To mediate their putative biological activity, lignans must first undergo absorption across the gastrointestinal barrier and subsequent delivery to the target tissues. Permeation across the intestinal epithelial barrier and conjugative metabolism within the intestinal epithelium are two important processes that can influence the oral bioavailability of lignans. Preliminary information concerning the intestinal permeation and phase II enzyme metabolism of lignans can be acquired using the Caco-2 cell culture model. Permeability estimates derived from this useful, predictive in vitro model of intestinal absorption and metabolism31,32 often correlate well with oral drug absorption characteristics in humans.33 This in vitro system also has been used to characterize the intestinal absorption and metabolism characteristics of other plant phenols such as the flavonoids and their glycosides.34,35 Hence, the purpose of this study was to evaluate the permeation characteristics of the plant-derived lignans, 1 and 2, and the mammalian lignans, 3 and 4, and to characterize the extent of phase II conjugative metabolism by the Caco-2 model. This information should provide insight into the oral bioavailability of each lignan and the possible bioactive lignan form associated with the health benefits of flaxseed lignan consumption.

Table 1. Permeation (Mean Papp ± SD) of Secoisolariciresinol Diglucoside (1), Secoisolariciresinol (2), Enterodiol (3), and Enterolactone (4) Transport across the Caco-2 Polarized Epithelium compound

Papp (A to B) (×10−6) cm/s

Papp (B to A) (×10−6) cm/s

EFRa

predicted Fac

1 2 3 4

BLQb 8.0 ± 0.4 7.7 ± 0.2 13.7 ± 0.2

BLQb 9.5 ± 0.4 8.9 ± 0.6 10.6 ± 0.5

1.2 1.2 0.8

0.90 0.90 0.94

a

The efflux ratio is defined as the quotient of the secretory permeability and the absorptive permeability [Papp,(B to A)/Papp(A to B)]. bBelow limit of quantification. cFormulas used in the prediction of Fa: Fa = 1 − (1 + 0.54 × Peff,man)−7 where log(Peff,man) = 0.4926 × log(Papp,Caco‑2,pH 7.4) − 0.1434.41,42

of 1 was limited, the maximum nontoxic lignan concentration (100 μM) was utilized as the final concentration in the wells to maximize the ability to make permeability assessments. Each lignan was tested for linear transport rates, and it was determined that transport was linear up to 2 h. A preliminary study showed that lignans are stable (>95%) in the test medium added to wells without cells and incubated for as long as 48 h. The apical-to-basolateral and basolateral-to-apical permeation rates for 1 could not be calculated as the concentrations were below the level of quantitation of the HPLC assay in the respective recipient compartments. These data suggest 1 demonstrates very poor permeation characteristics across the Caco-2 monolayer. The findings are consistent with the failure of in vivo studies to detect 1 in blood or urine and with studies of other polyphenolic glycosides,36 where transport across the intestinal epithelium was limited by poor passive diffusion characteristics and a lack of substrate specificity for the various uptake transporters expressed in the intestinal epithelium. The transepithelial transfer of 2−4 in the Caco-2 cell monolayer system was observed, and calculated efflux ratios of 2−4 were in the range 0.8−1.2 (Table 1). The apparent permeability coefficients of 2−4 were 30−50% of the apparent permeability of metoprolol ((29.9 ± 3.2) × 10−6 cm/s), a marker compound that separates compounds into high and low permeability classes in the biopharmaceutical classification system.37 The lignan apparent permeability coefficients suggest these lignans fall within the low permeability class of



RESULTS AND DISCUSSION Flaxseed contains significant levels of the lignan, 1, which is known to undergo conversion to its aglycone, 2, and further biotransformation to 3 and 4 in the gastrointestinal tract following oral consumption.20 In the literature, blood, urine, and tissue levels of 2−4, which exist largely as glucuronide or sulfate conjugates, have been reported, but not of 1.22−29 The 30

dx.doi.org/10.1021/np4004905 | J. Nat. Prod. 2014, 77, 29−34

Journal of Natural Products

Article

Figure 1. Time course of total (closed circle), free (open circle), and conjugated (triangle) (determined after enzymatic hydrolysis using βglucuronidase/sulfatase type H-5 from Helix pomatia) secoisolariciresinol diglucoside (1), secoisolariciresinol (2), enterodiol (3), and enterolactone (4) in incubation medium of Caco-2 cells grown on Transwell permeable inserts. Each lignan (100 μM) was added in triplicate to 24-well plates, and conjugation monitored up to 48 h. The data points are means ± SD and are expressed as a fraction of the initial lignan amount (100 μM) added to the Caco-2 cell monolayer at the beginning of the experiment.

metabolites 2−4 have the potential for complete absorption from the gastrointestinal tract in the absence of presystemic metabolism. Metabolites (2−4) of Secoisolariciresinol Diglucoside (1), But Not 1, Undergo Extensive Phase II Enzyme Metabolism in Caco-2 Monolayers. The extent of lignan glucuronidation and sulfation in the Caco-2 monolayer was assessed by evaluating the time course of lignan conjugation up to 48 h. Less than 3% of the total amount of 1 added to Caco-2 cells was conjugated by 48 h of incubation (Figure 1). Furthermore, treatment of cellular lysate and supernatant media with β-glucuronidase/sulfatase enzyme resulted in less than 5% conversion of 1 into 2. These data suggest 1 does not undergo phase II metabolism in intestinal cells and that the βglucuronidase/sulfatase enzyme utilized in this study is unable to cleave the glucose groups of 1 to yield the aglycone, 2. For compounds 2−4, glucuronidase/sulfatase-sensitive (phase II) conjugates were detected up to 48 h (Figure 1). Compounds 2 and 3 exhibited 95% and 90% conjugation in 48 h, respectively, while 4 was completely conjugated within 12 h. The initial rate of conjugation was higher for 4 as compared with 2 and 3. In this study the extent of conjugation of 3 and 4 in Caco-2 cells compared well with the values published by Jansen et al.43 Compounds 2−4 were extensively conjugated after 48 h, while 1 remained virtually unmetabolized in the Caco-2 system.

compounds. With calculated efflux ratios of 0.8−1.2, the present data also suggest that passive diffusion is the principal mechanism responsible for the transport of these lignans across the intestinal epithelium.38 The data obtained are consistent with During et al., who also reported that simple diffusion is the principal mechanism of lignan intestinal permeation.39 Nonetheless, these efflux values do not rule out the possible involvement of ATP binding cassette (ABC) or solute carrier (SLC) transporters in the uptake or efflux of lignans by the gastrointestinal tract mucosal barrier. ABC and SLC transporter expression in Caco-2 cells demonstrate important differences from expression levels in the small intestine and colon and, therefore, may not be predictive of carrier-mediated transport processes in vivo.40 Consequently, confirmation of the role of intestinal epithelial transporters in lignan oral absorption would require assessments in the intact gastrointestinal tract.34 Although the apparent permeability coefficients of 2−4 suggest these lignans exhibit low to moderate permeability at the intestinal barrier, the predicted fraction of their absorbed dose (Fa) in the intestine by the compartmental absorption and transit (CAT) model was more than 90% (Table 1). The CAT model incorporates intestinal transit time, apparent permeability, and radius of the small intestine to predict the fraction absorbed. Therefore, a comparison based on Fa is more appropriate than simple apparent permeability coefficients.41,42 Given Fa values >90% as predicted by the CAT model, the 31

dx.doi.org/10.1021/np4004905 | J. Nat. Prod. 2014, 77, 29−34

Journal of Natural Products

Article

consumption coupled with the present knowledge of their extensive phase II metabolism may warrant an investigation into the possible pharmacological activity of these conjugated metabolites.

These data suggest intestinal cells contribute to presystemic metabolism of 2−4. Although no study has specifically evaluated the absolute oral bioavailability of 1−4, the literature suggests their oral bioavailability is very low, and when present, they exist principally as conjugates of glucuronic acid. The data from this study suggest that the poor bioavailability of 2−4 is likely due to extensive presystemic metabolism and not due to their inability to permeate across the gastrointestinal tract. The data in this study further suggest the intestine likely makes an important contribution to presystemic metabolism. This is supported by Axelson et al., who detected glucuronide conjugates of 3 and 4 in the portal vein after the oral administration of 1 in rats.44 As well, glucuronide and sulfate conjugates of 3 and 4 have been detected in rhesus monkey and human hepatocytes and in human serum and urine following oral lignan administration.28,45 The lack of detectable levels of 1 in blood and tissues following oral consumption is likely due to the inability of 1 to permeate the gastrointestinal barrier. An inability to permeate the cell membrane also would explain the limited metabolism of 1, as this lignan would not become accessible to cytosolic β-glucosidases and intracellular conjugative enzymes. Interestingly, an absence of 1 metabolism also may suggest that brush border membrane β-glucosidases have no role in the hydrolysis of this compound. Caco-2 cells are known to express brush border membrane β-glucosidases albeit at low expression levels.46 The extent of conjugation by these lignans corresponded to the order of their increasing lipophilicity (1 < 2 < 3 < 4). The literature also reports a similar extent of conjugation.39 Since metabolic enzymes are located intracellularly, compounds must cross the apical membrane of enterocytes to undergo phase II metabolism. Compound 4 exhibited the highest apparent permeability and predicted absorbed fraction, which may explain in part its greater extent of conjugation in the Caco-2 monolayer. These data are similar to the uptake and metabolism characteristics of compound 4 reported in HepG2 liver cancer cell lines.47 Conversely, the poor permeability characteristics of 1 across the Caco-2 cell monolayer are consistent with its poor conjugative metabolism. In the absence of human in vivo absolute bioavailability studies, the use of in vitro human intestinal cell cultures can provide supportive evidence for the role of the intestine in presystemic metabolism. Although Caco-2 monolayers are not truly representative of the intestinal epithelium, their advantage over microsomal systems or S9 fractions prepared from epithelial scrapings is that compounds must first permeate the cell membrane to gain access to the intracellular enzymes involved in phase II metabolism. On the basis of the evaluations with the Caco-2 cell monolayer, this study may conclude that the principal lignan of flaxseed, 1, undergoes very limited intestinal cell permeation and metabolism and is not likely to be systemically available following oral administration. The metabolites, 2−4, though, passively permeate across the intestinal epithelium and exhibit appreciable conjugative metabolism. The ability of the Caco-2 cell monolayer to extensively metabolize 2−4 indicates a role for the intestine in the presystemic metabolism of these lignans. Further studies are required to determine the oral bioavailability and the exact contributions of the intestine and liver to presystemic metabolism. Furthermore, glucuronide conjugates are pharmacologically inactive with a few exceptions,48 but the compelling evidence of health benefits associated with flaxseed lignan



EXPERIMENTAL SECTION

General Experimental Procedures. Compounds 1 and 2 (>95% purity) were kind gifts from Agriculture and Agri-Food Canada, Saskatoon (Dr. Alister Muir). β-Glucuronidase/sulfatase type H-5 from Helix pomatia (G-1512), compounds 3 and 4 [>95% purity (HPLC)], and sodium acetate were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada). Caco-2 human colon adenocarcinoma cells were obtained as a kind gift from Dr. Wolfgang Koester (Vaccine and Infectious Disease Organization, University of Saskatchewan, Canada). Dulbecco’s modified Eagle’s medium (DMEM), Transwell plates, polyethylene inserts (6.5 mm diameter, 0.4 μm pore size), and 24-well tissue culture plates were purchased from Fisher Scientific (Toronto, ON, Canada). Phosphate-buffered saline (PBS), fetal bovine serum (FBS), trypsin, versene, and nonessential amino acids (NEAA) were obtained from Invitrogen (Burlington, ON, Canada). Umbelliferone (7-hydroxycoumarin) and riboflavin were purchased from Sigma-Aldrich Canada. HPLC grade acetonitrile was purchased from Fisher Scientific. Methanol was purchased from Caledon Laboratories (Georgetown, ON, Canada). All other chemicals unless mentioned were obtained from Sigma-Aldrich Canada. A Milli-Q Synthesis water purification system (Millipore, Bedford, MA, USA) provided purified deionized water. All chemicals used were of analytical grade. A Millicell ERS system for the measurement of transepithelial electrical resistance (TEER) values was purchased from Millipore (Billerica, MA, USA). Cell Culture Conditions. Caco-2 cells were used as a model for the small intestinal mucosal epithelium to investigate the permeation and metabolism of flaxseed (1 and 2) and mammalian (3 and 4) lignans. Caco-2 cells with passage numbers 41−54 were grown in DMEM containing 10% FBS and 1% NEAA. Cells were grown at 37 °C under 95% O2 and 5% CO2. Caco-2 cells were subcultured using 0.25% trypsin in versene for cellular detachment, and Caco-2 cells (1 × 105 cells/well) were seeded on polyester membrane Transwells (PET) and 24-well tissue culture plates for permeability and phase II conjugative metabolism studies, respectively. The medium was replaced in apical and basal compartments of the Transwells (PET) (Corning Life Sciences, 24-well format, 6.5 mm diameter, pore size of 0.4 μm and pore density of 4 × 106 pores/cm2, membrane thickness of 1 μm) and 24-well plates three times per week. Monolayers were formed on polyester membrane Transwells. Experiments with Caco-2 cells, except the cytotoxicity assay, were conducted after 21 days in culture. Cytotoxicity Assay. Cytotoxicity of flaxseed (1 and 2) and mammalian lignans (3 and 4) in Caco-2 cells was determined using the sulforhodamine B assay.49 Caco-2 cells were grown in T-75 flasks using a suitable growth medium, which was replaced three times in a week [growth area of 75 cm2, vented caps and straight neck, Fisher Scientific (Toronto, ON, Canada)]. Trypsin (0.25% in versene) and trypan blue dye were used to harvest and count the cells in an exponential phase. Complete medium (100 μL) containing about 5 × 103 cells was transferred into each well of 96-well plates, and cells were allowed to attach and grow for 24 h. After 24 h, complete medium (100 μL) containing different lignans (10−500 μM) (concentrations that can be attained within the colon of the gastrointestinal tract following oral consumption of flaxseed)50 in HBSS and control (1% DMSO) was added to cell-containing medium and incubated for 72 h. After 72 h, trichloroacetic acid (50 μL of 50% w/v in water) was added to each well, and wells were placed at 4 °C for 1 h for fixation of the cells. One 96-well plate was fixed at the start of the experiment to determine the number of cells present in the 96-well plates at the beginning of the experiment (Tz). Plates were washed with tap water four times and allowed to air-dry overnight. Staining of the cells was done using sulforhodamine (0.4% w/v in 1% v/v acetic acid) and washed with acetic acid (1% v/v). Trizma base (200 μL of 10 mM) 32

dx.doi.org/10.1021/np4004905 | J. Nat. Prod. 2014, 77, 29−34

Journal of Natural Products

Article

snail Helix pomatia was used for enzymatic hydrolysis of samples after incubating for different time points up to 48 h. Then 100 μL of 0.1 M sodium acetate buffer (pH 5.0) containing 0.66 mg of β-glucuronidase and sulfatase H-5 was added to 100 μL of sample. Samples were mixed briefly and incubated at 37 °C for 2 h. Samples were then cooled to room temperature, and 200 μL of acetonitrile was added. Samples were vortex-mixed for 5 min and centrifuged at 2300g for 10 min. Supernatant was transferred to centrifuge tubes, filtered through 0.2 μm filters, and analyzed by HPLC. Results are presented as a percentage of lignan as free (before enzymatic hydrolysis) and total (after enzymatic hydrolysis) relative to a zero time control. HPLC Analysis. Samples from the permeability and conjugative metabolism experiments were analyzed using an HPLC method as reported previously.51 Briefly, for 1 HPLC analysis, 10 μL of riboflavin (internal standard) (25 μg/mL) was added to 100 μL of calibration standards, quality control samples, and samples from the permeability (both apical and basolateral) and conjugative metabolism assays and vortex-mixed. For the HPLC analysis of compounds 2−4, 10 μL of umbelliferone (internal standard) (100 μg/mL) was added to all samples as described for 1 above. Samples (100 μL) were transferred to HPLC vials, and 50 μL of sample was injected onto the column. HPLC mobile phase conditions for 1 were different from those for 2− 4 as reported,40 but both involved gradient mobile phase conditions using acetonitrile (containing 0.1% formic acid) and water (containing 0.1% formic acid) at a flow rate of 1 mL/min. An excitation wavelength of 217 nm and an emission wavelength of 677 nm were used; the total run time was 25 min. Standard curves were constructed from 0.01 to 10 μg/mL for compounds 2 and 3 and 0.05 to 10 μg/mL for compounds 1 and 4 using weighted linear regression (with 1/X2 weighting factor). Statistical Analysis. All experiments had a minimum of three independent observations for each test group. Data were expressed as means ± SD where applicable. Comparison of means between two groups was performed using independent Student’s t test. Comparison of means between more than two groups was performed using oneway analysis of variance (ANOVA) along with Dunnett’s post hoc test. A Student t test was used to determine whether slopes and intercepts of the calibration curves were significantly different from zero using Prism 4.0 (GraphPad Prism, San Diego, CA, USA). The level of significance was set at p < 0.05. The graphs were drawn using base package in R version 2.13.1 (R Foundation for Statistical Computing, Vienna, Austria; ISBN 3-900051-07-0, http://www.R-project.org/).52

was added to dried wells, and absorbance was read at 515 nm. The percent growth was calculated using eq 1, where optical density (OD) indicates absorption. The IC50 was derived by fitting four-parameter logistic curves (nonlinear regression analysis) between percent cell growth and log concentration using GraphPad Prism 5.0 for Windows (GraphPad Software, San Diego, CA, USA). %cell growth =

ODsample − ODTz ODcontrol − ODTz

× 100

(1)

Lignan Permeability Assay. A stock solution (10 mM) of compound 1 was prepared in Hank’s balanced salt solution (HBSS), and stock solutions (10 mM) of compounds 2−4 were individually prepared using 1% DMSO in HBSS for permeability studies. Stock solution preparation and dilutions were performed under sterile conditions. On the day of the permeability assessment, cells were washed three times with HBSS (100 μL) with 10 mM HEPES buffer, pH 7.4. Monolayers were equilibrated at 37 °C for 30 min before measurement of TEER values of each well using the Millicell ERS system. Working stock (100 μM) solutions of 1−4 were added separately to apical (200 μL) or basolateral (600 μL) compartments of the Transwells and further incubated at 37 °C, 95% O2, and 5% CO2 for 2 h. At the end of the incubation period, samples were collected from both compartments and stored at −20 °C until further HPLC analysis. After sample collection, cells were again washed three times with HBSS containing 10 mM HEPES (pH 7.4) and equilibrated with this solution for 30 min before measurement of postassay TEER. Lucifer yellow (a marker for paracellular flux) in HBSS was added to the apical compartment of each Transwell, which were then incubated under the same conditions for 1 h. Fluorescence levels in the basal compartment (excitation 485 nm and emission 535 nm) were determined using a Biotek Synergy HT microplate reader (Fisher Scientific, Nepean, ON, Canada). The Lucifer yellow rejection rates and postassay TEER values were used as the parameters to ensure monolayer integrity during the experiment. Apparent permeability coefficients (Papp) and efflux ratios (EFR) were calculated for apical to basolateral (A to B) and basolateral to apical (B to A) directions following quantitative determination of lignans by HPLC. Calculations are based on the following equations as described: Papp =

EFR =

dQ 1 × Am × dt C0



(2)

Papp(B − A) Papp(A − B)

Corresponding Author

(3)

*Tel: +1 306 966 6365. Fax: +1 306 966 6377. E-mail: jane. [email protected].

where dQ/dt = rate of permeation, Am = surface area of monolayer, and C0 = initial concentration in donor compartment. In vivo intestinal permeability in humans (Peff,man) was calculated using eq 4,41 and fraction of drug absorbed (Fa) was predicted using eq 5.42

log(Peff,man) = 0.4296 × log(Papp,Caco ‐ 2,pH7.4) − 0.1434

(4)

Fa = 1 − (1 + 0.54 × Peff,man)−7

(5)

AUTHOR INFORMATION

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This project was made possible through a contribution from the Rexall Research Trust Fund, College of Pharmacy and Nutrition, University of Saskatchewan. J.K.M. was funded by a University of Saskatchewan graduate student scholarship.

Conjugation of Lignans in Caco-2 Cells. After 21 days of culture, Caco-2 cells were washed three times with HBSS containing 10 mM HEPES buffer, pH 7.4. Stock solutions (10 mM) and working solutions (100 μM) of compounds 1−4 were prepared in DMEM. Each lignan working stock solution (700 μL) was added in triplicate to the 24-well plates and incubated at 37 °C, 95% O2, and 5% CO2 for 48 h. Supernatant and cell pellets were collected at 0, 2, 4, 6, 12, 24, and 48 h in triplicate from 24-well plates. Supernatant was transferred to 2 mL polypropylene centrifuge tubes. Cold PBS (4 °C, 200 μL) was added to the cell layer, and culture plates were subsequently scraped with a rubber policeman to collect residual cells. Cell suspensions were centrifuged at 76g (Eppendorf Centrifuge 5804 R, Mississauga, ON, Canada) at 4 °C for 5 min. Following centrifugation, cells were stored at −20 °C without disturbing cell pellets until enzymatic hydrolysis. A preparation containing both β-glucuronidase and sulfatase from the



REFERENCES

(1) Pandey, K. B.; Rizvi, S. I. Oxid. Med. Cell Longev. 2009, 2, 270− 278. (2) Peterson, J.; Dwyer, J.; Adlercreutz, H.; Scalbert, A.; Jacques, P.; McCullough, M. L. Nutr. Rev. 2010, 68, 571−603. (3) Bergman Jungeström, M.; Thompson, L. U.; Dabrosin, C. Clin. Cancer Res. 2007, 13, 1061−1067. (4) Thompson, L. U. Baillieres Clin. Endocrinol. Metab. 1998, 12, 691−705. (5) Johnsson, P.; Kamal-Eldin, A.; Lundgren, L. N.; Aman, P. J. Agric. Food Chem. 2000, 48, 5216−5219.

33

dx.doi.org/10.1021/np4004905 | J. Nat. Prod. 2014, 77, 29−34

Journal of Natural Products

Article

(6) Felmlee, M. A.; Woo, G.; Simko, E.; Krol, E. S.; Muir, A. D.; Alcorn, J. Br. J. Nutr. 2009, 102, 361−369. (7) Zhang, W.; Wang, X.; Liu, Y.; Tian, H.; Flickinger, B.; Empie, M. W.; Sun, S. Z. Br. J. Nutr. 2008, 99, 1301−1309. (8) Prasad, K. Atherosclerosis 2008, 197, 34−42. (9) Prasad, K. Atherosclerosis 2005, 179, 269−275. (10) Prasad, K.; Mantha, S. V.; Muir, A. D.; Westcott, N. D. Atherosclerosis 1998, 136, 367−375. (11) Prasad, K. J. Lab Clin. Med. 2001, 138, 32−39. (12) Pan, A.; Demark-Wahnefried, W.; Ye, X.; Yu, Z.; Li, H.; Qi, Q.; Sun, J.; Chen, Y.; Chen, X.; Liu, Y.; Lin, X. Br. J. Nutr. 2009, 101, 1145−1149. (13) Pan, A.; Sun, J.; Chen, Y.; Ye, X.; Li, H.; Yu, Z.; Wang, Y.; Gu, W.; Zhang, X.; Chen, X.; Demark-Wahnefried, W.; Liu, Y.; Lin, X. PLoS One 2007, 2, e1148. (14) Thompson, L. U.; Chen, J. M.; Li, T.; Strasser-Weippl, K.; Goss, P. E. Clin. Cancer Res. 2005, 11, 3828−3835. (15) Tan, K. P.; Chen, J.; Ward, W. E.; Thompson, L. U. Exp. Biol. Med. 2004, 229, 147−157. (16) Chen, L. H.; Fang, J.; Li, H.; Demark-Wahnefried, W.; Lin, X. Mol. Cancer Ther. 2007, 6, 2581−2590. (17) Zhang, W.; Wang, X.; Liu, Y.; Tian, H.; Flickinger, B.; Empie, M. W.; Sun, S. Z. J. Med. Food. 2008, 11, 207−214. (18) Jenab, M.; Thompson, L. U. Carcinogenesis 1996, 17, 1343− 1348. (19) Kamal-Eldin, A.; Peerlkamp, N.; Johnsson, P.; Andersson, R.; Andersson, R. E.; Lundgren, L. N.; Aman, P. Phytochemistry 2001, 58, 587−590. (20) Nesbitt, P. D.; Lam, Y.; Thompson, L. U. Am. J. Clin. Nutr. 1999, 69, 549−555. (21) Morton, M. S.; Chan, P. S.; Cheng, C.; Blacklock, N.; MatosFerreira, A.; Abranches-Monteiro, L.; Correia, R.; Lloyd, S.; Griffiths, K. Prostate 1997, 32, 122−128. (22) Kuijsten, A.; Arts, I. C.; Vree, T. B.; Hollman, P. C. J. Nutr. 2005, 135, 795−801. (23) Rickard, S. E.; Thompson, L. U. J. Nutr. 1998, 128, 615−623. (24) Niemeyer, H. B.; Honig, D. M.; Kulling, S. E.; Metzler, M. J. Agric. Food Chem. 2003, 51, 6317−6325. (25) Smeds, A. I.; Saarinen, N. M.; Hurmerinta, T. T.; Penttinen, P. E.; Sjöholm, R. E.; Mäkelä, S. I. J. Chromatogr. B. 2004, 813, 303−312. (26) Saarinen, N. M.; Huovinen, R.; Wärri, A.; Mäkelä, S. I.; Valentín-Blasini, L.; Sjöholm, R.; Ammälä, J.; Lehtilä, R.; Eckerman, C.; Collan, Y. U.; Santti, R. S. Mol. Cancer Ther. 2002, 1, 869−876. (27) Kurzer, M. S.; Lampe, J. W.; Martini, M. C.; Adlercreutz, H. Cancer Epidemiol. Biomarkers Prev. 1995, 4, 353−358. (28) Dean, B.; Chang, S.; Doss, G. A.; King, C.; Thomas, P. E. Arch. Biochem. Biophys. 2004, 429, 244−251. (29) Adlercreutz, H.; van der Wildt, J.; Kinzel, J.; Attalla, H.; Wähälä, K.; Mäkelä, T.; Hase, T.; Fotsis, T. J. Steroid Biochem. Mol. Biol. 1995, 52, 97−103. (30) Niemeyer, H. B.; Metzler, M. J. Chromatogr. B 2002, 777, 321− 327. (31) Shah, P.; Jogani, V.; Bagchi, T.; Misra, A. Biotechnol. Prog. 2006, 22, 186−198. (32) Artursson, P.; Borchardt, R. T. Pharm. Res. 1997, 14, 1655− 1658. (33) Artursson, P.; Karlsson, J. Biochem. Biophys. Res. Commun. 1991, 175, 880−885. (34) Teng, Z.; Yuan, C.; Zhang, F.; Huan, M.; Cao, W.; Li, K.; Yang, J.; Cao, D.; Zhou, S.; Mei, Q. PLoS One 2012, 7, e29647. (35) Murota, K.; Shimizu, S.; Miyamoto, S.; Izumi, T.; Obata, A.; Kikuchi, M.; Terao, J. J. Nutr. 2002, 132, 1956−1961. (36) Liu, Y.; Hu, M. Drug Metab. Dispos. 2002, 30, 370−377. (37) Volpe, D. A. AAPS J. 2004, 6, 1−6. (38) Sun, H.; Chow, E. C.; Liu, S.; Du, Y.; Pang, K. S. Expert. Opin. Drug Metab. Toxicol. 2008, 4, 395−411. (39) During, A.; Debouche, C.; Raas, T.; Larondelle, Y. J. Nutr. 2012, 142, 1798−805.

(40) Hilgendorf, C.; Ahlin, G.; Seithel, A.; Artursson, P.; Ungell, A. L.; Karlsson, J. Drug Metab. Dispos. 2007, 35, 1333−1340. (41) Sun, D.; Lennernas, H.; Welage, L. S.; Barnett, J. L.; Landowski, C. P.; Foster, D.; Fleisher, D.; Lee, K. D.; Amidon, G. L. Pharm. Res. 2002, 19, 1400−1416. (42) Yu, L. X.; Amidon, G. L. Int. J. Pharm. 1999, 186, 119−125. (43) Jansen, G. H.; Arts, I. C.; Nielen, M. W.; Müller, M.; Hollman, P. C.; Keijer, J. Arch. Biochem. Biophys. 2005, 435, 74−82. (44) Axelson, M.; Setchell, K. D. FEBS Lett. 1981, 123, 337−342. (45) Knust, U.; Hull, W. E.; Spiegelhalder, B.; Bartsch, H.; Strowitzki, T.; Owen, R. W. Food Chem. Toxicol. 2006, 44, 1038−1049. (46) Chantret, I.; Rodolosse, A.; Barbat, A.; Dussaulx, E.; BrotLaroche, E.; Zweibaum, A.; Rousset, M. J. Cell. Sci. 1994, 107 (Part 1), 213−225. (47) Adlercreutz, H.; Mousavi, Y.; Clark, J.; Höckerstedt, K.; Hämäläinen, E.; Wähälä, K.; Mäkelä, T.; Hase, T. J. Steroid Biochem. Mol. Biol. 1992, 41, 331−337. (48) Paul, D.; Standifer, K. M.; Inturrisi, C. E.; Pasternak, G. W. J. Pharmacol. Exp. Ther. 1989, 251, 477−483. (49) Vichai, V.; Kirtikara, K. Nat. Protoc. 2006, 1, 1112−1116. (50) Hu, C.; Yuan, Y. V.; Kitts, D. D. Food Chem. Toxicol. 2007, 45, 2219−2227. (51) Mukker, J. K.; Kotlyarova, V.; Singh, R. S.; Alcorn, J. J. Chromatogr. B 2010, 878, 3076−3082. (52) RCoreTeam. R: A Language and Environment for Statistical Computing; R Foundation for Statistical Computing: Vienna, Austria, 2012, http://www.R-project.org/.

34

dx.doi.org/10.1021/np4004905 | J. Nat. Prod. 2014, 77, 29−34