Pharmacological assessment of horse chestnut seed extracts and its major constituent aescin in Caco-2 cell monolayer cultures Spanakis M ., Niopas I., Vizirianakis I.S. Department of Pharmacognosy & Pharmacology, School of Pharmacy, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece Corresponding author: Vizirianakis IS, Laboratory of Pharmacology, Department of Pharmacology & Pharmacognosy School of Pharmacy, AUTH, e-mail: ivizir@pharm.auth.gr Abstract Nowadays, it is of great importance for pharmacology to predict drug response by applying validated assays, either early in new drug development process, or in the clinical settings. In an effort to establish an infrastructure in our laboratory to clinically assess drug interactions, we were able to confirm that herbal medicinal products of horse chestnut seed extracts (HCSE) inhibit drug metabolizing enzymes CYP3A4 and CYP2D6 in vitro. In this work, we applied the Caco-2 cell model system used in drug absorption studies to further investigate the pharmacological relevance of these observations. In particular, we evaluated the effect of HCSE and its constituent aescin in Caco-2 cells by assessing: a) The viability of cells through the application of MTT assay; b) The cellular integrity of membranes by measuring transepithelial electric resistance (TEER); and c) The adherent junctions’ morphology. The data obtained thus far indicate that HCSE and aescin: 1) Do not significantly affect cell viability; 2) Alter cellular integrity as seen by TEER values; Interestingly, TEER exhibited an initial significant reduction within the first 3hr of treatment that subsequently has been reversed leading thereafter to a substantial increase by 24hr; and 3) Modulate E-cadherin levels at cellular junctions. Overall, these data propose that the potential alteration of cell membrane integrity by HCSE and aescin could affect the paracellular drug transport in the intestine, although such a conclusion needs further pharmacological and clinical investigation Reference list Methodology Results 0 4000 8000 12000 16000 20000 0 12 24 36 48 Cells /well Time (hr) Control Horse Chestnut 0,005 mg/ml Horse Chestnut 0,050 mg/ml Horse Chestnut 0,250 mg/ml Horse Chestnut 0,500 mg/ml 0 4000 8000 12000 16000 20000 0 12 24 36 48 Cells /well Time (hr) Control Aescin 0.1 μΜ Aescin 0.5 μΜ Aescin 1 μΜ Aescin 5 μΜ Horse chestnut seed extract Aescin Immunofluorescence assays:  The immunofluorescence experiments revealed a potential modulation of Caco-2 cell layers organization after 48 hr exposure in HCSE. Similar results were also obtained with aescin.  Both the HCSE and aescin seemed to influence E-cadherin localization in cell adherens junctions, whereas there is minimal effect on desmoglein-2 and β-catenin levels in Caco-2 cells.  The changes in cell membrane level of E-cadherin might be also correlated with the modulation in transepithelial resistance observed after 3 and 24 hr exposure in HCSE- and/or aescin-treated Caco-2 cells.  MTT :  No significant cytotoxicity was observed for the tested HCSE concentrations after 48 h exposure of Caco-2 cell cultures.  Similarly, small cytotoxicity was observed in the higher concentrations of aescin (5μΜ) tested. In particular, approximately 30% reduction in cell growth after 48 h incubation was observed.  TEER values:  A significant decrease in the TEER values has been seen at the early stages of the application of either HCSE or aescin, whereas after 24 hr the phenomenon was reversed and the TEER values were increased again reaching initial values even in the presence of HCSE and aescin.  The modulation of TEER values was shown to follow a similar trend in both HCSE- and/or aescin-exposed cells. The latter, tends to propose that the effect on TEER values is attributed to aescin. 25% 50% 75% 100% 125% 150% 0 3 6 9 12 15 18 21 24 % TEER Time (hr) Control HCSE 0.25 mg/ml Aescin 0.1 μM 1. Abebe, W. (2002). "Herbal medication: potential for adverse interactions with analgesic drugs." J Clin Pharm Ther 27(6): 391-401. 2. Guney, G., H. M. Kutlu and A. Iscan (2012) "The Apoptotic Effects of Escin in The H-Ras Transformed 5RP7 Cell Line." Phytother Res. doi: 10.1002/ptr.4804. 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Vascul Pharmacol 49(4-6): 158-65. 1 st assay  Incubation of Caco-2 cells in 96 well plate in various concentrations of HCSE and aescin and estimation of non-toxic concentrations up to 48 hours.  Cytotoxicity was performed using the MTT assay. 2 nd assay  Estimation of any potential modulation of cellular integrity and the formation of epithelial cell layers.  Measurement of Transepithelial Electric Resistance in a non-toxic concentration for 24 hr in Caco-2 cell cultures grown in suitable “Transwells inserts”. 3 rd assay  Immunofluorescence experiments to assess cell-cell adhesion contacts in order to evaluate potential alteration of adherens and/or desmosomal junctions in Caco-2 cell cultures.  immunofluorescence was performed to assess the level of E-cadherin, desmoglein-2 and β-catenin adhesion molecules. 9-11 November 2012 Heraklion, Crete, Greece, Foundation for Research and Technology Caco-2 cell model represents a clinically-validated system applied upon new drug development to study drug intestine permeability as well as to assess drug interactions in vitro. Upon an effort to evaluate potential modulation of drug absorption process in patients also taking the herbal medicinal product HCSE, Caco-2 cells were used to assess cytotoxicity, cell morphology as well as cell membrane integrity and permeability. Moreover, the effect of aescin that represents the major constituent of HCSE was simultaneously assessed to allow better understanding of the data taken. The three assays applied are shown below. Aim Discussion The assessment of herbal medicinal products (HMPs) is very important in order to predict and evaluate their pharmacological actions in the body as well as to better clarify to which compounds of HMPs any specific pharmacological actions can be attributed. This study demonstrates that HCSE may partially modify the cellular morphology of Caco-2 cells and this effect might be attributed to aescin which is the major ingredient of the herb with known anti-inflammatory properties. The changes observed in transmembrane electrical resistance and the morphology in cell adherence junctions of the cells as shown by the TEER and immunofluorescence experiments indicate that potential modulation could be observed in absorption of drugs through the gastrointestinal tract in the presence of HCSE and/or aescin. These preliminary data for HCSE need further pharmacological and clinical investigation in order to more thoroughly estimate the clinical significance of the results taken. On the other hand, however, the present study clearly shows that, upon conducting studies in the era of drug-herb interactions, the application in vitro of methodologies already validated and used in new drug development process as well as upon assessment of drug-drug interactions could be useful and informative. To this end, such a direction also opens new ways for clinically evaluating the effects of HMPs in everyday practice.