S. wallichii Korth. leaves were collected from Lembang, West Java, Indonesia. The plant species was identified at the Department of Biology, Faculty of Mathematics and Natural Sciences, University of Padjadjaran (West Java, Indonesia).

The S. wallichii leaves were dried and extracted with 70% ethanol at room temperature three times for 24 h each. A concentrated extract was obtained in vacuo at 50°C. The ethanol extract was partitioned into n-hexane, ethyl acetate and aqueous phases. Column chromatography on a Wakogel C 200 (Wako Pure Chemical Industries, Ltd., Osaka, Japan) column was performed to the ethyl acetate fraction, as it was previously reported as the most active fraction against cancer cell lines, using a mixture of n-hexane, ethyl acetate and methanol with increasing polarity. The major compound observed was purified using silica G 60 with sulfuric acid ethanol (1:9) and was found to be the most active fraction of S. wallichii, which was characterized and analyzed as described previously (4). The isolate was, however, identified by spectroscopic methods (ultraviolet, infrared and nuclear magnetic resonance) and liquid chromatography mass spectrometry (5).

The LNCaP human breast cancer cell line was purchased from Dainippon Pharmaceutical (Tokyo, Japan). The non-cancerous esophageal cell line (CHEK-1, an immortalized human esophageal cell line) was provided by Dr H. Matsubara. CHEK-1 was established by the transduction of the human papillomavirus type 16 E6/E7 into primary cultures of esophageal keratinocytes (6). The cell lines were cultured in RPMI-1640 medium (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). For the cell treatments, various concentrations of kaempferol-3-O-rhamnoside were added to the cell culture medium for 24 h, at which time the medium was replaced. The cells were subsequently collected at the indicated times.

A cell proliferation analysis was performed in the presence of various concentrations of kaempferol-3-O-rhamnoside using a colorimetric MTT assay, as described in a previous study (7). Briefly, the cells were plated in 96-well plates (2×10⁴ in 50 µl/well). Following the initial cell seeding, the indicated concentrations of extract were applied and incubated for 24 h. WST-8 assay cell-counting solution (10 µl) (Dojindo Lab., Tokyo, Japan) was added to each well and incubated at 37°C for 3 h. After the addition of 1 M HCl (100 µl/well), the cell proliferation rates were determined by measuring the absorbance at a wavelength of 450 nm with a reference wavelength of 650 nm using a microtiter plate reader (Becton-Dickinson, Franklin Lakes, NJ, USA). The results were derived from triplicate experiments.

Protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA). Proteins (40 µg) were electrophoresed on 5–20% Tris-Tricine ReadyGel (Bio-Rad, Tokyo, Japan) and electro-transferred to a Hybond-enhanced chemiluminescence membrane (Amersham, Buckinghamshire, UK). Apoptosis-related proteins were analyzed by immunoblot analysis using caspase-3 (cat no. 9668), caspase-8 (cat no. 9746), caspase-9 (cat no. 9508) and poly (ADP-ribose) polymerase (PARP) antibodies (cat no. 9542) at a 1:1,000 dilution (Cell Signaling Technology, Beverly, MA, USA). β-actin (cat no. A1978; Sigma) served as the loading control.

Based on a previous study (4), it was found that the major compound of the ethyl acetate fraction of the S. wallichii extract was kaempferol-3-O-rhamnoside. The compound was purified, isolated and identified as kaempferol-3-O-rhamnoside (C₂₁H₂₀O₁₀ or 3,4′,5,7-tetrahydroxyflavone-3-O-rhamnoside) with a molecular weight of 432 (Fig. 1).

The effect of kaempferol-3-O-rhamnoside on the viability of LNCaP and CHEK-1 cells was evaluated. The result of the MTT assay indicated that the treatment of cancer (LNCaP) and non-cancer (CHEK-1) cell lines with kaempferol-3-O-rhamnoside resulted in dose-dependent inhibition of cell growth (Fig. 2). Twenty-four hours of treatment with kaempferol-3-O-rhamnoside inhibited the proliferation of the LNCaP cells with a half maximal inhibitory concentration (IC₅₀) value of 218 µM. However, the inhibition of the CHEK-1 cell proliferation with kaempferol-3-O-rhamnoside demonstrated an IC₅₀ of 386 µM, which was higher than that of the LNCaP cells. This may indicate that kaempferol-3-O-rhamnoside has fewer cytotoxic effects on the non-cancerous cells.

Kaempferol-3-O-rhamnoside treatment of the LNCaP cells induced the upregulation of caspase-8, caspase-9 and caspase-3 in a time-dependent manner (Fig. 3). Furthermore, the changes in the expression levels of PARP, a hallmark biomarker of apoptosis, suggested that kaempferol-3-O-rhamnoside may induce apoptosis on LNCaP cells through the activation of caspase-dependent signaling pathways after 24 h of treatment.