396 Anti-human immunodeficiency virus activity of 3,4,5-tricaffeoylquinic acid in cultured cells of lettuce leaves Hirotoshi Tamura 1 , Takashi Akioka 1 , Koichi Ueno 1 , Takeshi Chujyo 1 , Katsu-ichiro Okazaki 2 , Peter J. King 3 and W. Edward Robinson, Jr. 3 1 Department of Biochemistry and Food Science, Kagawa University, Miki-cho, Kagawa, Japan 2 Departmet of Life Science, Kagawa University, Miki-cho, Kagawa, Japan 3 Department of Pathology, University of California, Irvine, CA, USA 3,4,5–Tricaffeoylquinic acid (TCQA) that is not found in intact plant of lettuce leaves was isolated from the cultured cells. The intact plant produced chicoric acid (dicaffeoyl tartaric acid: L-CCA) as well as chlorogenic acid (3-caffeoylquinic acid: 3-CQA) as the major metabolites. After subculturing of the cells for 40 days, the amount of 3,4,5-TCQA reached to 0.14 mg/g fresh weight. The inhibitory effect of 3,4,5-TCQA for human immunodeficiency virus (HIV) Type 1 integrase was assayed. Anti- HIV activity using HIV and MT-2 cells was 1.15 lM and IC 50 against HIV integrase was 0.063 lM whereas cell toxicity of this chemical was expressed as 5% death of all living cells to be 18.4 lM. The HIV inhibitory effect of 3,4,5-TCQA was the highest in values among L-CCA, and other dicaf- feoylquinic acids. This datawill provide a new possibility for creating a new drug design for HIV. Keywords: Cultured cell / Integrase / Lactuca sativa / Lettuce / Tricaffeoylquinic acid / Received: October 21, 2005; revised: December 4, 2005; accepted: December 5, 2005 DOI 10.1002/mnfr.200500216 Mol. Nutr. Food Res. 2006, 50, 396 – 400 1 Introduction Human immunodeficiency virus (HIV)/AIDS has been an epidemic of 40 million people at the end of 2003. HIV-1 integrase plays a crucial role in the HIV-1 virus replication cycle. An essential step in the viral life cycle is integration of the viral DNA into the host genome (Fig. 1). It catalyzes to snip the host DNA and also to incorporate virus genome to the snipped ends. Human cells do not have any needs to cut-and-paste DNA fragments into their genome. There- fore, the inhibition of integrase should be a good target for drug therapy, since it is not likely to interfere with the nor- mal operation of human cells [1]. Robinson et al. [2] comparatively reported selective inhibi- tory effects of dicaffeoylquinic acids (DCQA) and chicoric acid (L-CCA) for HIV-1 integrase. Synthetic approach for seeking new HIV integrase inhibitors was also tried among caffeoyl derivatives [3, 4]. Cultured cells from certain plants may also give some possibility of providing the target drugs all seasons. So, we would like to report herein that 3,4,5-tricaffeoylqui- nic acid (TCQA) from cultured cells of the lettuce (Lactuca sativa var. crispa L.) would be a potent inhibitor against HIV-1 integrase among mono-, di-, and tricaffeoyl deriv- atives. 2 Materials and methods 2.1 HPLC analysis of 3-caffeoylquinic acid (3-CQA) and the related compounds 3-CQA and the related compounds were analyzed by an HPLC instrument (980-PU, JASCO) under the gradient elu- tion with a mixed solvent of AcOH : CH 3 CN:H 2 O:H 3 PO 4 = 20 : 25 : 53.5 : 1.5 (this solvent system is expressed as 100%- P) and 1.5% H 3 PO 4 aqueous solution at 1.0 mL/min. Meta- bolites were detected at 325 nm (UV-975, JASCO). The chromatography was effectively conducted by using a 5 lm Develosil Ph-HG-5 column, 250 mm64.6 mm id. During the initial 5 min, 40%-P solvent was eluted and then the sol- vent was gradually changed to 100%-P for 20 min. Finally, the 100% P solvent was kept flowing for 10 min. Correspondence: Professor Hirotoshi Tamura, Department of Bio- chemistry and Food Science, Kagawa University, Miki-cho, Kagawa, 761-0795 Japan E-mail: tamura@ag.kagawa-u.ac.jp Fax:+81-87891-3021 Abbreviations: 3-CQA, 3-caffeoylquinic acid; DCQA, dicaffeoylqui- nic acids; HIV, human immunodeficiency virus; 3,4,5-TCQA, 3,4,5- tricaffeoylquinic acid i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com