Caco-2 6. Caco-2. colon adenocarcinoma cell line NEAA Sigma-Aldrich. Caco-2. Transwell #3414 COL 6 Transwell # cm 2 75 cm 2 Caco-2

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1 * μg cm h 6 6 human 1. 2 DMEMDulbecco's modified eagle's medium FBS colon adenocarcinoma cell line 1 NEAA PBS Gibco DMSO Ⅰ RTC Sigma-Aldrich Transwell #3414 COL 6 Transwell # cm 2 75 cm 2 Corning Costar J. T. Baker 1. 3 Galaxy B CO 2 RS Biotech Evom World Precision rat tail collagen RTC STIK Instrument Sarasota FL XDS-1 GRX-9051B 6 HZS-H ATCC #HTB-37 ATCCAmerican type culture collection ZX BAI07B08 * Tel bjmu. edu. cn ADMET / Act. yangyanfang@ bjmu. edu. cn TGL-16C HPLC LC- 10AT vp SPD-M10A vp Class-vp DMEM PBS HBSSHanks' Balanced Salts Solution Transwell 515

2 0. 5 g L μg cm - 2 coefficientsp 1 app cm 2 P app = dq /dt 1 /A 1 /C 0 cm s ml 6 Transwell 1 h 2 h 1 ml PBS Table 1 2 Transwell Characteristics of two kinds of transwell plates #3414 # mm cm 2 3 μm DMEM-20 3 DMEM-10 4 ~ 5 d % C R 2 = A = 3 775C + DMEM / R 2 = ~ 120 μmol ml 6 BL 2. 6 ml DMEM-10 6 AP 1. 5 ml 2-3 AP 1. 5 ml BL 2. 6 ml 40 ~ TEER = R cell - R filter A Ω cm 2 apparent permeability Q μg dq /dt μg min C 0 μg L A cm 2 # HPLC Dikma Diamonsil C mm 250 mm 5 μm Dikma Easy Guard C mm 20 mm 20 μl 1 ml min nm -0. 2% % HBSS A = L P app cm s cm s transepithelium electrical resistanceteer 4 TEER R cell Ω R filter Ω A cm d 2 12 Transwell 0. 5 ml HPLC 1. 5 ml 6 516

3 2 n = 6 Table 2 TEER values of monolayer grown under different situationsn = 6 Ω cm 2 /h /μg cm - 2 /d ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 61 COL ± ± ± ± ± ± 67 1 P < P < A. HBSSB. C. 1 Fig HPLC HPLC chromatograms of propranolol 1and atenolol ml 2 517

4 3 Table 3 P app n = 3 P app values of propranolol and atenolol in monolayer grown under different situations n = 3 /h /μg cm - 2 P app / 10-5 cm s - 1 / 10-7 cm s ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± COL ± ± J J P app P app 3 J P app 10-7 cm s #3414 COL Transwell # μg cm h 6 J. J Chin Pharm Sci J. Int J Pharm HPLC-UV DAD J Yang X WTeng JXu Wet al. The permeability and the efflux of alkaloids of the Evodiae Fructus in the model J. Phytother Res J J Zhang L QHuang Y DYang X W. Intestinal permeability of liquiritin and isoliquiritin in the cell monolayer model 9 Tian X JYang X WYang X Det al. Studies of intestinal permeability of 36 flavonoids using cell monolayer model 10Yang X WHuang XMa Let al. The intestinal permeability of neolignans from the seeds of Myristica fragrans in the cell monolayer model J. Planta Med J

5 Wu QZhao BYang X Wet al. Intestinal permeability of ses- 12. quiterpenes in the cell monolayer model J. Planta Med J J. J Ethnopharmacol Zhao BYang X BYang X W. Intestinal permeability of the constituents from the roots of Saposhnikovia divaricata in the human cell monolayer modelj. Planta Med Wang YYang X W. Intestinal transport of free anthraquinones in cell model J. Chin J Nat Med J J Wu QYang X W. The constituents of Cibotium barometz and their permeability in the human monolayer cell model J. 19. J Yang X WTeng JZhao Bet al. Studies on absorption and transport of limoninoids from Fructus Evodiae in cell monolayer model J. Chin Herb Med Establishment of cell monolayer model with collagen coating 6-well plates for study of traditional Chinese medicine prescription YANG Yan-fangWU NiYANG Xiu-wei * Department of Natural MedicinesState Key Laboratory of Natural and Biomimetic Drugs School of Pharmaceutical SciencesPeking UniversityBeijing China Abstract cell monolayer model is widely utilized in drug absorption study and 12-well transwell TM plates were commonly used to study the absorption of different kinds of natural products. To establish a stable method for the study of traditional Chinese medicine prescription6-well plates were chosen because of the larger well volumes than 12-well plates. To study the impacts of collagen kindscoating density as well as coating time on the cell culture the transepithelial electrical resistance of cell monolayers grown on different collagen coating transwells was determinedand the permeations of propranolol and atenolol as standard markers were detected with HPLC. The results showed that the kinds of collagen the different coating densities and coating time of rat tail collagen had no significant influences on the cell monolayer integrality and absorption capacity. 6-well plates coated with 2 μg cm - 2 rat tail collagen for 1 hour were enough reliable and suitable for the study of traditional Chinese medicine prescription in vitro. Key words cell monolayer6-well platescollagentransepithelium electrical resistanceapparent permeability coefficients doi /cjcmm