Journal of Biochemistry and Molecular Biology, Vol. 36, No. 4, July 2003, pp. 387-389 © KSBMB & Springer-Verlag 2003 The Effect of Willow Leaf Extracts on Human Leukemic Cells in Vitro Hany A. El-Shemy †,§, *, Ahmed M. Aboul-Enein † , Mostafa I. Aboul-Enein ‡ , Sohair I. Issa ‡ and Kounosuke Fujita § † Department of Biochemistry, Faculty of Agriculture, Cairo University, Giza, Egypt ‡ Department of Clinical Pathology, National Cancer Institute, Cairo University, Cairo, Egypt § Faculty of Applied Biological Science, Hiroshima University, Hiroshima 739-8528, Japan Received 5 January 2003, Accepted 25 February 2003 The young developing leaves of willow (Salix safsaf, Salicaceae) trees have antileukemic activity. After a 24-h incubation in vitro, the crude water extracts of the leaves killed a majority of the blasts of acute myeloid leukemia (AML, 73.8%). Keywords: Acute myeloid leukemia, Human leukemic cell, Willow leaf Introduction The genus Salix contains about 300 species in the world. A screening program was initiated by Leven et al. (1979), that identified the antibacterial and antifungal activities as well as the antiviral, antiparasitic, and other pharmacologically active substance activities in higher plants. The extracts of certain plants are known to yield active antimicrobial substances which have been documented as phytochemicals of the genus of the family, and reported as the toxicity of the plants. Many components that are derived from medical and dietary plants possess potential chemopreventive properties (Han et al., 2002). To our knowledge, however, there has been no research concerning its effects on tumor cells. Therefore, this work investigated the effect of willow leaf extracts on the viability of leukemic cells. Materials and Methods The willow leaves (S. safsaf) were collected from the Salix farm of the Faculty of Agriculture, Cairo University, Giza, Egypt. The young leaves were directly extracted with hot water (7% concentration, 7 g of fresh leaves boiled (100 o C) in 100 ml distilled. water for 20 min, then filtered through a sterilized Miracloth and centrifuged at 15,000 rpm for 15 min). The solvent extraction was carried out with the plant leaves as follows: 80 g leaves were extracted consecutively at room temperature with petroleum ether (40-60 o C), that was followed by diethyl ether, chloroform, acetone, and finally with 70% ethanol. The solvent of each extract was removed by distillation at a low temperature, and each class of crude plant extract was separated for the next study. The study was performed on adult leukemic patients, aged 18- 65 years, that were admitted to the National Cancer Institute, Cairo University. The healthy volunteers (6 samples) and patients that included 15 AML (acute myeloid leukemia, immature monocytes) were diagnosed by peripheral blood and bone marrow examination, cytochemistry, and immunological markers, when needed. The healthy volunteers and patients were subjected to separation of mononuclear cells done by Ficoll hypaque density gradient (Pharmacia, Uppsala, Sweden). The cells were then washed three times with PBS and the counts were adjusted to 10 5 cells/0.1 ml (both mature and immature cells). The culture medium was prepared using modified Earles-salt with 1.2 g/l sodium carbonate and L-glutamine (Gibco, Grand island, USA), 10% inactivated fetal bovine serum (Gibco), and penicillin/ streptomycin was added. The medium was then filtered through 0.22 μm Millipore filters, one ml of which was transferred into a 1.8-ml screw-capped sterile plastic tube. Next, 0.1 ml of the cell suspension containing 10 5 cells was added to 5 tubes. To three of these tubes, 0.1 ml of the willow extract was added, while the other two tubes served as negative and positive controls. Culture medium was used instead of the willow extract for the negative control and the willow extract was added in the cells from healthy volunteers as a positive control. The tubes were incubated at 37 o C in the presence of 5% CO 2 for 24 h. The cells were tested for their viability using the trypan blue exclusion test (Bennett et al., 1976). Two hundred cells were counted, then the percentage of viable cells was estimated. *To whom correspondence should be addressed. Tel: 20-2-7742-600; Fax: 20-2-5717-355 E-mail: helshemy@hotmail.com