Biosynthesized tea polyphenols showed antichlamydial activity against Chlamydia trachomatis D/UW-3/Cx and L 2 /434/Bu using cell culture. The most active compounds were (؊)-epigallocatechin gallate and (؊)-epicatechin gallate, followed by (؊)-epicatechin (EC). (؉)-Epicatechin and (؊)-epigallocatechin were intermediate. EC was the least toxic. These results warrant evaluation of tea polyphenols as topical antichlamydial agents.Chlamydia trachomatis is one of the most common causes of sexually transmitted diseases (5). Results obtained with topical antichlamydial agents such as nonoxynol-9 (3, 8, 10), monocaprin (4), C31G (14), cecropin peptides (1), and protegrins (17) have been reported. Disinfectants containing products such as chlorhexidine gluconate gel (9) and the spermicide benzalkonium chloride (2) have also been shown to inactivate C. trachomatis. We have previously reported that tea extracts have antichlamydial activity in vitro and that the active compounds are polyphenols (16).Five tea polyphenols ( Fig. 1), (ϩ)-catechin (Catech), (Ϫ)-epicatechin (EC), (Ϫ)-epigallocatechin (EGC), (Ϫ)-epicatechin gallate (ECg), and (Ϫ)-epigallocatechin gallate (EGCg), were tested in this study. These chemicals were biosynthesized naturally and supplied by Mitsui Norin Co., Ltd. (Tokyo, Japan).The chlamydial strains tested were C. trachomatis D/UW-3/Cx and L 2 /434/Bu. HeLa 229 cells were used for growing C. trachomatis. A preinoculation minimal cidal concentration method, previously described by Lampe et al. (9), was used to test the in vitro susceptibility of C. trachomatis to tea polyphenols. A total of 1 ml of culture medium containing 2 ϫ 10 5 HeLa 229 cells per ml was dispensed into each well of a plastic 24-well culture plate and incubated in 5% CO 2 at 37°C for 24 h to form a confluent monolayer. Then, 1.0 ϫ 10 4 inclusionforming units of C. trachomatis were incubated with serial dilutions of tea polyphenols at 35°C for 90 min. Controls were incubated with sucrose phosphate glutamate (SPG) buffer containing no test compounds. Pretreated inocula were centrifuged onto the cells at 1,500 rpm for 60 min. After centrifugation, the inocula were removed. Culture medium containing no tea polyphenols was added and incubated for 72 h. Eagle's minimum essential medium containing 10% fetal calf serum and 0.6 g/ml cycloheximide was used as the culture medium. The cells were fixed with methanol and stained with fluorescein isothiocyanate-conjugated antichlamydial monoclonal antibody (Denka Seiken Co., Ltd., Tokyo, Japan). Inclusions were counted by a fluorescent microscope. At least three wells per dilution were tested, and each experiment was repeated at least three times.The toxic effects of EC, ECg, and EGCg on HeLa 229 cells were examined by using a CK01 cell counting kit (Dojindo Laboratory Co., Ltd., Kumamoto, Japan), a colorimetric assay for cell proliferation and viability. Cell activity was determined according to the manufacturer's instructions. Briefly, serial dilutions of tea polyphenols in SPG buffer were dispense...