Send Orders for Reprints to reprints@benthamscience.ae 36 Open Chemistry Journal, 2015, 2, 36-39 1874-8422/15 2015 Bentham Open Open Access Flavonoids from the Fresh Leaves of Kalanchoe tomentosa (Crassulaceae) Lilis Siti Aisyah 1 , Yenny Febriany Yun 1 , Euis Julaeha 1 , Tati Herlina 1 , Achmad Zainuddin 1 , Wawan Hermawan 2 , Unang Supratman 1,* and Hideo Hayashi 3 1 Department of Chemistry, Faculty of Mathematics and Natural Sciences, Padjadjaran University, Jatinangor 45363, Sumedang, Indonesia; 2 Department of Biology, Faculty of Mathematics and Natural Sciences, Padjadjaran University, Jatinangor 45363, Sumedang, Indonesia; 3 Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Gakuen-cho, Sakai, Osaka 599-8531, Japan Abstract: Flavonoids compounds such as kaempferol (1), kaempferol-3-O--D-glucopyranoside or astragalin (2) and kaempferol-3-O--L-rhamnoside or afzelin (3) have been isolated from the fresh leaves of Kalanchoe tomentosa (Crassulaceae). The chemical structure of isolated compounds 1-3 were identified by spectroscopic evidences and comparison with those compound previously reported. Compounds 1-3 showed cytotoxic activity against P-388 murine leukimia cells with IC 50 values of 51.8,  100 and 3.32 μg/mL, respectively. Keywords: Crassulaceae, cytotoxic activity, flavonoids, Kalanchoe tomentosa, P-388 murine leukimia cells. INTRODUCTION Kalanchoe tomentosa (Crassulaceae) is a perennial, succulent medicinal herb which grown in high terrain and indigenous to low altitude of Indonesia [1]. The leaves of K. tomentosa are used in Indonesian folk medicine for the treatment of fever, infections, rheumatism and skin diseases [2]. The genus of Kalanchoe reported to contain bufadienolides [3-5], triterpenoids [6] and flavonoids [7-9] which possess multiplebiological activities such as blocking human lymphocyte proliferation [6,10], cytotoxic agents [11], insecticidal properties [5, 12] and inhibiting cancer cell growth [13, 14]. However, information about phytochemical constituents of K. tomentosa is unavailable. Our preliminary screening for novel cytotoxic agents from Indonesian Kalanchoe plants, we found that the methanolic extract of K. tomentosa exhibited significant cytotoxic effect against P- 388 murine leukimia cells. In this communication, the isolation and structure identification of flavonoids (1-3) along with their cytotoxic activity against P-388 murine leukimia cells will be described. EXPERIMENTAL General Experimental Procedures Ultra-Violet spectra were recorded in methanol on Jasco UV-1575 spectrophotometer. The IR spectra were measured on a Perkin Elmer spectrum-100 FT-IR in KBr. Mass spectra were obtained with a Water, Qtof HR-MS XEV otm mass spectrometer. NMR spectra were recorded with a JEOL JNM A-500 spectrometer using tetra methyl silane (TMS) as an internal standard. Chromatographic separation was done on *Address correspondence to this author at the Department of Chemistry, Faculty of Mathematics and Natural Sciences, Padjadjaran University, Jl. Raya Bandung-Sumedang Km 21, Jatinangor 45363, Sumedang, West Java, Indonesia; Tel/Fax: +62-22-7794391; E-mail: u_supratman@unpad.ac.id silica gel 60 (Merck). PTLC glass plates were precoated with silica gel GF 254 (Merck, 0.25 mm). TLC plates were precoated with silica gel GF 254 (Merck, 0.25 mm) and detection was achieved with 10% H 2 SO 4 in ethanol followed by heating. Plant Material The fresh leaves of K. tomentosa were collected from Lembang Discrict, West Bandung, Indonesia in May, 2011. The plant was identified in Bogoriense Herbarium, Bogor, Indonesia and a voucher specimen (No. B0-129211) was deposited at the herbarium. Extraction and Isolation Fresh grounded leaves (20 Kg) of K. tomentosa were extracted with MeOH at room temperature. The MeOH extract was evaporated under reduced pressure to yield a dark brown residue (360 g). The MeOH extract was dissolved in water and partitioned succesively with n- hexane, EtOAc and n-butanol. Evaporation of each solvents resulted in the crude extract of n-hexane (30.5 g), EtOAc (64.5 g) and n-butanol (43.5 g), respectively. The n-hexane, ethyl acetate and n-butanol extracts exhibited a cytotoxic activity against P-388 murine leukimia cells with IC 50 values of 56.5, 24.4 and 45.2 μg/mL, respectively. A portion of the EtOAc extract (50 g) was subjected to vacuum liquid chromatography on silica gel G60 using gradient elution of n-hexane-EtOAc-MeOH to afford 15 fractions (A01-A015). Fraction A05 (3.5 g) was further subjected to column chromatography on silica gel (70-230 mesh) using mixture of n-hexane-EtOAc (10:0-5:1) as eluting solvents to afford 10 fractions (B01-B10). Fraction B04 (230 mg) was subjected to flash column chromatography on silica gel (230- 400 mesh), eluted with CHCl 3 -MeOH (9:1), to give 1 (12.4 mg). Fraction A06-A07 was combined (4.2 g) and subjected