Evaluation of Intestinal Permeability of Vicenin-2 and Lychnopholic

Article pubs.acs.org/jnp

Evaluation of Intestinal Permeability of Vicenin‑2 and Lychnopholic Acid from Lychnophora salicifolia (Brazilian Arnicão) Using Caco‑2 Cells Dayana Rubio Gouvea,† Arthur de Barros Bello Ribeiro,† Ursula Thormann,‡ Norberto Peporine Lopes,† and Veronika Butterweck*,‡ †

NPPNS (Núcleo de Pesquisa em Produtos Naturais e Sintéticos), Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14040-903 Ribeirão Preto-SP, Brazil ‡ School of Life Sciences, Institute for Pharma Technology, University of Applied Sciences Northwestern Switzerland, CH-4132 Muttenz, Switzerland

ABSTRACT: Lychnophora salicifolia, commonly known as “arnicão”, is used as an anti-inflammatory agent and as a flavoring agent in the Brazilian traditional spirit “cachaça”. In this work, the permeation process of vicenin-2 (1) and lychnopholic acid (2) (major secondary metabolites from the hydroalcoholic extract) was investigated using Caco-2 cells. For this investigation, a new HPLC-DAD method was developed and validated for the quantification step. It was observed that 2 crosses the Caco-2 cell monolayer by passive diffusion. On the other hand, 1 was not transported, suggesting no absorption and no efflux of this compound in Caco-2 cells.

L

ychnophora salicifolia Mart. (Asteraceae, Vernonieae) is an endemic plant from the Brazilian Cerrado known as “arnicão”. The leaves of Lychnopora spp. are used as flavorings for the Brazilian traditional spirit “cachaça”.1 The major constituents of the hydroalcoholic extracts prepared from the leaves of L. salicifolia are vicenin-2 (1) and lychnopholic acid (2).2 Compound 1 is a C-glucoside flavonoid that has been shown to be effective as an anti-inflammatory3 and an antioxidant4 and also exhibits anticancer effects.5 It was further shown that lychnopholic acid is responsible for the antibacterial6 and trypanocidal activities7 of L. salicifolia.

the absorption process by the use of controlled conditions, and the reduction of the use of animals, which often can be controversial, expensive, and time-consuming.8 However, one of the most appealing attributes that these experimental systems possess is their capability to perform a relatively high-throughput screening procedure. Caco-2 cells are human colon adenocarcinoma cells, able to differentiate and polarize when grown spontaneously in inserts, forming a monolayer with tight junctions. It is possible to correlate the permeation of the compounds in this system with the characteristics of permeation in the human intestinal mucosa, so that they can be used to predict oral drug absorption in humans and are also useful in studies of the mechanism of the absorption of new substances.9 Herbal medicines are multicomponent mixtures that contain several active compounds; thus, the determination of pharmacokinetic parameters is relatively complex. However, the pharmacological importance of vicenin-2 (1) and lychnopholic Special Issue: Special Issue in Honor of Otto Sticher

In vitro ADME studies using cell culture models have several advantages such as time efficiency, the possibility of monitoring © 2013 American Chemical Society and American Society of Pharmacognosy

Received: August 19, 2013 Published: November 26, 2013 464

dx.doi.org/10.1021/np400674t | J. Nat. Prod. 2014, 77, 464−471

Journal of Natural Products

Article

Figure 1. Chromatograms obtained from HPLC-DAD analysis (A) at 254 nm, lychnopholic acid (2); (B) 325 nm, vicenin-2 (1); (C,D) 254 and 325 nm, apigenin.

validation of the method are shown in Table 1. Each compound was monitored at its maximum absorption wavelength. The apparent permeability (Papp) value of lychnopholic acid (2) was calculated from the results obtained in the experiment performed with the transport medium at pH 7.4 in the apical compartment and in the basolateral compartment at the concentrations of 50 and 25 μM of 2 (Figure 2). The values obtained for two different concentrations (50 and 25 μM) were very similar, suggesting no involvement of active transport. The results are presented below: 50 μM: Papp A→B = 4.07 × 10−6 cm/s; Papp B→A = 4.17 × 10−6 cm/s; Papp A→B/Papp B→A = 0.97, and Papp B→A/Papp A→B = 1.02.

acid (2) depends on their availability for intestinal absorption and subsequent interaction with target tissues. Thus, it was the aim of the present study to quantify and to characterize the in vitro transport of 1 and 2 using human intestinal epithelial Caco2 cells in order to obtain further insight into their absorption mechanisms.

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RESULTS AND DISCUSSION The analytical methodology was validated in accordance with current international guidelines.10 Figure 1 is an example of a chromatogram obtained from HPLC-DAD analysis for the standards of vicenin-2 (1) and lychnopholic acid (2), and the results of the 465



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Table 1. Values Obtained for the Validation of the Analytical Methodology for the Quantification of Vicenin-2 (1) and Lychnopholic Acid (2)a,b parameter evaluated

values obtained

detection limit (μM) superior quantification limit (μM) inferior quantification limit (μM) concentrations evaluated using culture medium (μM)

a

vicenin-2 (1)

lychnopholic acid (2)

1.75 400 3.15 50

120 0.86 1.15 0.97 0.00 106

25

0.59 0.18 1.27 0.74 102

100

30

3.75 480 7.5 60

0.46 0.31 0.79 0.61 102

3.10 1.87 2.06 0.18 111

0.76 1.79 1.23 0.01 108

repeatability RSD (%) intermediate precision RSD (%) within day accuracy RE (%) between days accuracy RE (%) recovery stability (%) 25 °C

0.30 2.04 9.97 9.58 108

24 h freezing cycles (%)

100

99

99

100

98

99

1x 2x 3x

101 101 100

100 100 100

100 100 100

99 98 97

97 99 98

99 100 100

RSD: relative standard deviation expressed as a percentage (%), n = 3. bRE: relative error expressed as a percentage (%), n = 3.

Figure 2. Distribution of lychnopholic acid (2) after transepithelial transport experiments using transport medium rich in glucose (25 mM). (A−D) At pH 7.4; (E,F) using pH 6.0 in the apical and pH 7.4 in the basolateral compartment. (A,E) Compound 2 (50 μM) placed in the apical compartment; (B,F) compound 2 (50 μM) placed in the basolateral compartment; (C) compound 2 (25 μM) placed in the apical compartment; and (D) compound 2 (25 μM) placed in the basolateral compartment.

25 μM: Papp A→B = 4.68 × 10−6 cm/s; Papp B→A = 4.41 × 10−6 cm/s; Papp A→B/Papp B→A = 1.06, and Papp B→A/Papp A→ B = 0.94. The ratio Papp B→A/Papp A→B being close to 1.0 suggests that the molecule crosses the Caco-2 cells monolayer by passive diffusion.11 When the pH of the apical compartment was changed from 7.4 to 6.0, which corresponds more closely to the intestinal pH, 2 crossed the monolayer with a much higher rapidity (Figure 2). When the compound was tested at pH 7.4 at the apical and basolateral compartments, 2 was detected at the basolateral compartment only 30 min after the beginning of the experiment. When the apical pH was changed from 7.4 to 6.0, 2 was detected after 5 min in the basolateral compartment. Based on the log D values calculated (at pH 6.0 = 1.24 and at pH 7.4 = −0.09), pH 6.0 favors the acidic form of the compound

(to remain in its nonionized form), thus increasing nonpolarity and thereby facilitating passive diffusion across the membrane. From these results, it was concluded that lychnopholic acid (2) permeates the cell monolayer by passive diffusion. However, the present results are in line with those reported for the transport of indomethacin (also an acidic compound) at different pH values.11b The authors have shown that the transport of indomethacin decreased with increased pH, as expected from the pH-partition hypothesis. Net absorption occurred when the basolateral pH exceeded the apical pH. It was also demonstrated that active and passive drug transport results were indistinguishable in temperature dependency studies.11b The permeation of vicenin-2 (1) in the Caco-2 cell model was not observed under any of the conditions tested. Diniz et al.12 used QRAR models based on the retention time in micellar 466



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Figure 3. Distribution of vicenin-2 (1) after transepithelial transport experiments using transport medium rich in glucose at pH 7.4. (A) Compound 1 (50 μM) placed in the apical compartment; (B) compound 1 (50 μM) placed in the basolateral compartment; (C) compound 1 (100 μM) placed in the apical compartment; and (D) compound 1 (100 μM) placed in the basolateral compartment.

transporter due to the high glucose concentration. Therefore, experiments were conducted under a low glucose concentration (5 mM), glucose-free transport medium and using HBSS as transport medium, but 1 still was not transported under these conditions (Figure 4). To investigate if the sugar has an influence on the permeation, apigenin, the aglycone of 1, was investigated. As shown in Figure 5, apigenin was absorbed by Caco-2 cells but could be detected neither in the basolateral compartment (“apical to basolateral direction”) nor in the experiments in the other direction (“basolateral to apical direction”). This can probably occur by cell accumulation, as previously reported by Tian et al.14 and because most of the absorbed apigenin can be metabolized by the Caco-2 cells.15 Considering the popular use of the leaves of Lychnopora spp. as flavorings for the Brazilian traditional spirit “cachaça”, it was evaluated if the permeation of lychnopholic acid (2) and vicenin2 (1) changes when provided in the form of an extract. Recently published data have shown that after oral administration of Abrus mollis extract in mice, 1 could be detected in plasma samples,16 but in this case, other compounds in the extract might have enhanced the absorption of the compound. Our results are shown in Figure 6, indicating no change in permeation profile of both compounds. Thus, lychnopholic acid (2) did not enhance the absorption of 1. However, as to how far other compounds might influence the permeation of 1 needs to be evaluated in further experiments. The toxicity of the isolated compounds was evaluated in Caco2 cells (Figure 7). The TEER value gives information about the integrity of the cell monolayer and about cell integrity and, therefore, about toxicity. Significant TEER changes indicate that the compound may be toxic to Caco-2 cells or that the cell

chromatography to estimate the oral absorption rate of 1 and found a negative value of log P (−4,31), which indicates high polarity and good water solubility, and therefore, a low absorption of this compound can be expected. The calculated log D values are also negative (log D at pH 7.4 = −4.36 and at pH 6.0 = −3.24). Nagaprashantha et al.5 provided the first data that the isolated compound in mice can be absorbed after oral administration. However, this does not reflect the conditions used in the present study because, in the earlier case, vicenin-2 was administered orally for 8 weeks.5 Although the present results showed that in Caco-2 cells this compound was not transported, this fact does not exclude the possibility of absorption of 1 in vivo under certain conditions, like the presence of other compounds in the extract (coeffectors) that can promote its absorption. Figure 3 shows that the amount of 1 in the “apical to basolateral direction” experiment remained constant and unchanged during all evaluated time points. The same observation was made when the apical pH was changed to 6.0 and the composition of the transport medium as well as the amount of 1 were kept constant at both compartments of the transwell plate. In the efflux experiments (from basolateral to apical), the amount of 1 did not change in the basolateral compartment and no vicenin-2 was detected in the apical compartment. Since the vicenin-2 molecule has two glucose units attached at C-6 and C-8, the possibility of active absorption via glucose transporters was considered. There are also reports in the literature of other glucosylated flavonoids that are absorbed by these transporters.13 As the initial experiments were conducted under a high concentration of glucose (25 mM), the observed results could be influenced by the saturation of the glucose 467



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Figure 4. Distribution of vicenin-2 (1) after transepithelial transport experiments. (A,B) Using transport medium low in glucose (5 mM) and pH 7.4; (C,D) using glucose-free transport medium and pH 7.4; (E,F) using glucose-free transport medium at pH 6.0 in the apical and 7.4 in the basolateral compartment; and (G,H) using HBSS as transport medium. (A,C,E,G) Compound 1 (50 μM) placed in the apical compartment; (B,D,F,H) compound 1 (50 μM) placed in the basolateral compartment.

Based on the present results, it is concluded that lychopholic acid (2) crosses the Caco-2 cells’ monolayer by passive diffusion. The compound showed good permeability in this model and might have a high absorption rate in vivo.17 The C-glucoside, vicenin-2 (1), did not cross the cell monolayer under the

monolayer suffered injury. On increasing the concentration of the compounds, no significant changes in the TEER values were observed when compared with the control (transport medium free of the compounds studied). This demonstrates that the compounds are not appreciably toxic to cells. 468



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Figure 5. Distribution of apigenin after transepithelial transport experiments using a glucose-free transport medium at pH 6.0 in the apical and pH 7.4 in the basolateral compartment. (A) Apigenin placed in the apical compartment, and (B) apigenin placed in the basolateral compartment, both at a concentration of 25 μM. (NPL 327) is deposited at the UEC Herbarium. After collection, plant material was brought to the laboratory and then dried, as soon as possible, at 40 °C under forced ventilation for 48 h. Extraction and Isolation. A dried powder of L. salicifolia leaves (400 g) was extracted exhaustively with 50% ethanol. The ethanol extract (40 g) was suspended in 90% methanol and partitioned with hexane. After this step, lychnopholic acid (2) was precipitated as a white powder (200 mg). The lychnopholic acid (2) was then washed with cold hexane and cold water. A part of the dried methanolic fraction (5 g) was chromatographed over Sephadex LH-20 (70 g), using isocratic solvent elution with methanol. Nine fractions were collected and pooled according to TLC examination to obtain one fraction containing vicenin-2 (1) as the principal component. This fraction was chromatographed by reversedphase preparative HPLC-DAD to obtain pure 1 (50 mg). Preparative chromatographic separation was performed on aShimadzu, ShimPack ODS column (5 μm, 250 mm × 20 mm) at room temperature. The HPLC-DAD system consisted of a Shimadzu LC-6AD apparatus equipped with a diode array detector (DAD) (SPD-M10Avp, Shimadzu, Tokyo, Japan), coupled with an autoinjector (SIL-10AF, Shimadzu, Tokyo, Japan). The mobile phase consisted of water (A) and methanol (B). The flow rate was 9 mL/min, and an injection volume of 1 mL was used. Elution profile: 0 to 30 min: 15 to 55% B (linear gradient), 30 to 35 min: 55 to 100% B (linear gradient), 35 to 40 min (column washing): 100% B (isocratic), 40 to 55 min: 100 to 15% B (linear gradient), 55 to 60 min (column equilibration): 15% B (isocratic). The UV-DAD detector was set to record between 220 and 600 nm, and UV chromatograms were recorded at 325 nm. To confirm compound identity, the retention time, the accurate mass, ESI fragmentation profile, and UV spectra of isolated substances were compared with data already published in the literature.2 The vicenin-2 (1) purity was 99% as determined by HPLC analysis, and for lychnopholic acid (2), the purity was 98%. HPLC-DAD Analysis Used for Evaluation of Compound Permeability in Caco-2 Cells. The analyses were performed on an Agilent HPLC-DAD (Agilent, Waldbronn, Germany) consisting of a DAD detector model (G4212A) (DEBAF00471), an autosampler model low flow hip sampler (G1377A) (DE64555676) and a cap pump (G1376a) (DEAAG00144). A Nucleodur Macherey-Nagel C18 gravity, 3 μm (70 mm × 4.0 mm) column (Machery-Nagel, Oensingen, Switzerland), and a mobile phase consisting of acetonitrile (B) and water (A), both containing 1% acetic acid, were used. The column was kept at room temperature, and the samples were conditioned at 25 °C in the autosampler. The flow rate was 0.3 mL/min, and an injection volume of 5 μL was used. Elution profile: 0 to 5 min: 5 to 45% B (linear gradient), 5 to 10 min: 45 to 100% B (linear gradient), 10 to 13 min: 100% B (column washing), 13 to 14 min: 100 to 5% B (linear gradient), 14 to 17 min: 5% B (isocratic, column equilibration). The UV-DAD detector was set to record between 210 and 600 nm, and UV chromatograms were recorded at 254 and 300 nm.

Figure 6. Distribution of vicenin-2 (1) and lychnopholic acid (2) after transepithelial transport experiments using a transport medium at pH 7.4. (A) Compounds 1 and 2 placed together in the apical compartment, and (B) compounds 1 and 2 placed together in the basolateral compartment, both at a concentration of 50 μM.

conditions studied. However, this fact does not exclude the possibility of absorption of vicenin-2 in vivo under certain conditions.

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EXPERIMENTAL SECTION

General Experimental Procedures. All materials used for cell culture were the same as previously described by Kapitza et al.18 Acetonitrile and acetic acid (HPLC grade) were purchased from Sigma (Buchs, Switzerland), and water was purified by a Arium pro Ultrapure water system (Sartorius Stedim Biotech GmbH, Goettingen, Germany). Apigenin (≥99% HPLC purity) and Hank’s balanced salt solution were purchased from Sigma-Aldrich (Sigma, Buchs, Switzerland). Plant Material. Leaves of Lychnophora salicifolia were collected in Piatã, Bahia-Brazil (S 13°06′11.3″ W 41°51′20.3″) and identified by Prof. Dr. João Semir (University of Campinas). A voucher specimen 469



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Figure 7. TEER values (Ω·cm2) over time utilizing transport medium pH 7.4 in the apical and basolateral compartments with differents concentrations of (A) vicenin-2 (1) and (B) lychnopholic acid (2). Evaluation of Intestinal Permeability of Vicenin-2 (1) and Lychnopholic Acid (2). The cells were cultured as reported by Kapitza et al.18 The integrity and transportation ability of the Caco-2 cell monolayer was examined by measuring the transepithelial electrical resistance (TEER) with an epithelial voltohmmeter before and after the experiments (World Precision Instruments, Sarasota, FL, USA). Prior to the experiment, the Caco-2 cell monolayer was washed with warm Dulbecco’s PBS (containing Ca2+ and Mg2+) and preincubated with transport medium for 1 h at 37 °C and under 8% CO2 before measuring the TEER. The monolayer was only used if their TEER values were between 400 and 650 Ω·cm2. For the permeation assays, solutions of the compounds were added to the apical side to evaluate the permeation from the apical to basolateral direction. To evaluate the permeation from the basolateral to apical side, the solutions were added to the basolateral side of the transwell plate. Passage of the compounds through the monolayer was monitored by taking samples (100 μL) from both apical and basolateral compartments. The volume was not replaced until the end of the experiment, before the final TEER measurement. To ensure the sink conditions, each time a sample from the apical compartment was withdrawn, the same volume was drawn from the basal compartment and vice versa. The initial volumes used were 1.6 mL in the apical and 2.8 mL in the basolateral compartment. During the experiment, the transwell plate was kept at 37 °C in a water-saturated atmosphere and under agitation with an orbital agitator at 120 rpm (Edmund Bühler, Hechingen, Germany). Samples were injected directly (5 μL injection volume) and analyzed by HPLC-DAD. Experiments were carried out in triplicate; the data represent mean ± SD. The following equation was used to calculate the apparent permeability coefficients, Papp = (ΔQ/Δt)/AC0, where ΔQ/Δt is the linear appearance rate of the compound on the receiver side (μM/s), A is the membrane surface area (cm2), and C0 is the initial concentration in the donor compartment (μM/cm3). Log D calculations were performed using the Marvin Sketch software (version 6.0.4, ChemAxon Ltd., Budapest, Hungary). The parameters used for this were as follows: static acid-basic prefix, macro mode, considering tautomerism and resonance, Cl−, Na+, K+ concentrations of 0.1 mol/dm3, and temperature of 25 °C.

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would also like to thank IBAMA (Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis) for License No. 029/2006 and CNPq (Conselho Nacional de Desenvolvimento ́ e Tecnológico) for authorizing access to a component Cientifico of genetic heritage (No. 010143/2011-4), and FAPESP (Fundaçaõ de Amparo à Pesquisa do Estado de São Paulo - proc. no. 2010/ 09137-0), CNPq, and CAPES for financial support.

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DEDICATION Dedicated to Prof. Dr. Otto Sticher, of ETH-Zurich, Zurich, Switzerland, for his pioneering work in pharmacognosy and phytochemistry.

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REFERENCES

(1) Semir, J.; Rezende, A. R.; Monge, M.; Lopes, N. P. As Arnicas Endêmicas das Serras do Brasil; UFOP Ed.: Ouro Preto, Brazil, 2011; p 212. (2) Gouvea, D. R.; Meloni, F.; de Barros Bello Ribeiro, A.; Lopes, J. L. C.; Lopes, N. P. Anal. Chim. Acta 2012, 748, 28−36. (3) Marrassini, C.; Davicino, R.; Acevedo, C.; Anesini, C.; Gorzalczany, S.; Ferraro, G. J. Nat. Prod. 2011, 74, 1503−1507. (4) Gobbo-Neto, L.; Santos, M. D.; Kanashiro, A.; Almeida, M. C.; Lucisano-Valim, Y. M.; Lopes, J. L. C.; Souza, G. E. P.; Lopes, N. P. Planta Med. 2005, 71, 3−6. (5) Nagaprashantha, L. D.; Vatsyayan, R.; Singhal, J.; Fast, S.; Roby, R.; Awasthi, S.; Singhal, S. S. Biochem. Pharmacol. 2011, 82, 1100−1109. (6) Miguel, O. G.; Lima, E. O.; Morais, V. M. F.; Gomes, S. T. A.; Delle Monache, F.; Cruz, A. B.; Cruz, R. C. B.; Cechinel, V. Phytother. Res. 1996, 10, 694−696. (7) Jordão, C. O.; Vichnewski, W.; De Souza, G. E. P.; Albuquerque, S.; Lopes, J. L. C. Phytother. Res. 2004, 18, 332−334. (8) Artursson, P. J. Pharm. Sci. 1990, 79, 476−482. (9) Wilson, G.; Hassan, I. F.; Dix, C. J.; Williamson, I.; Shah, R.; Mackay, M.; Artursson, P. J. Controlled Release 1990, 11, 25−40. (10) ICH Q2(R1) Validation of Analytical Procedures: Definitions and Terminology; International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, Geneva, Switzerland, 1996. (11) (a) Krishna, G.; Chen, K. W. J.; Lin, C. C.; Nomeir, A. A. Int. J. Pharm. 2001, 222, 77−89. (b) Neuhoff, S.; Ungell, A. L.; Zamora, I.; Artursson, P. Eur. J. Pharm. Sci. 2005, 25, 211−220. (12) Diniz, A.; Escuder-Gilabert, L.; Lopes, N. P.; Gobbo-Neto, L.; Villanueva-Camanas, R. M.; Sagrado, S.; Medina-Hernandez, M. J. J. Agric. Food Chem. 2007, 55, 8372−8379. (13) (a) Gee, J. M.; DuPont, M. S.; Rhodes, M. J. C.; Johnson, I. T. Free Radical Biol. Med. 1998, 25, 19−25. (b) Walgren, R. A.; Lin, J. T.; Kinne, R. K.; Walle, T. J. Pharmacol. Exp. Ther. 2000, 294, 837−843. (14) Tian, X. J.; Yang, X. W.; Yang, X. D.; Wang, K. Int. J. Pharm. 2009, 367, 58−64.

AUTHOR INFORMATION

Corresponding Author

*Tel: +41 61 467 46 89. Fax: +41 61 467 47 01. E-mail: veronika. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge Prof. Dr. João Semir (Instituto de Biologia, Universidade Estadual de CampinasUNICAMP, Brazil) for identification of the plant. The authors 470



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(15) Hu, M.; Chen, J.; Lin, H. M. J. Pharmacol. Exp. Ther. 2003, 307, 314−321. (16) Wang, H.; Jiang, Z.; Du, H.; Liang, C.; Wang, Y.; Zhang, M.; Zhang, L.; Ye, W.; Li, P. J. Chromatogr. B 2012, 903, 68−74. (17) Artursson, P.; Karlsson, J. Biochem. Biophys. Res. Commun. 1991, 175, 880−885. (18) Kapitza, S. B.; Michel, B. R.; van Hoogevest, P.; Leigh, M. L. S.; Imanidis, G. Eur. J. Pharma. Biopharm. 2007, 66, 146−158.

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Evaluation of Intestinal Permeability of Vicenin-2 and Lychnopholic

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