β-Sitosterol Oxides From the Dried Stem of Salix gilgiana Inhibit the Proliferation of HL-60 Leukemic Cells

Background: Salix gilgiana is a deciduous tree that grows in northern Japan, the Korean peninsula, eastern Russia along the Ussuri River, and northeast China. The stem of this tree is dried and consumed orally as a folk medicine. Our intensive screening of various plant materials found that the MeOH extract of its dried stem exhibited significant antiproliferative activity against HL-60 leukemic cells with an IC50 of 16 ppm. We systematically investigated the biologically active compounds of the MeOH extract of the dried stem of S. gilgiana. Methods: The MeOH extract of S. gilgiana dried stem was fractionated by a repeated chromatography monitored by antiproliferative activity against HL-60 leukemic cells. Five active compounds were isolated and the structures were elucidated by MS, 1H- and 13C-NMR spectroscopy, and X-ray analysis. Results: The active compounds were identified as 7-ketositosterol (I), 7β-hydroxysitosterol (II), 7α- hydroxysitosterol (III), (4-hydroxyphenyl)ethanol (IV), and (4-hydroxyphenyl)propan-1-ol (V). The strongest activity was found for 7α-hydroxysitosterol (III), with an IC50 of 8.4 µM. This is the first report of the isolation of these compounds from S. gilgiana. Conclusion: Five compounds were isolated by a repeated chromatography under the guidance of antiproliferative bioassay using HL-60. The structures were identified as three β-sitosterol oxides and two phenolic compounds. Since Salix species, namely, willow trees, have beneficial characteristics including rapid growth, easy cloning, and resistance to high humidity and dryness, they may be utilized as a relatively inexpensive tool for the efficient production of useful bioactive materials.

Comparison of the cytotoxic effects of β-sitosterol oxides and a cholesterol oxide, 7β-hydroxycholesterol, in cultured mammalian cells

Phytosterols are plant sterols found in foods such as oils, nuts and vegetables. Phytosterols, in the same way as cholesterol, contain a double bond and are susceptible to oxidation. The objective of the present study was to assess the potential toxic effects of β-sitosterol oxides on U937 cells. The effects of increasing concentrations (0-120 μM) of β-sitosterol oxides on cellular cytotoxicity, apoptosis, anti-oxidant status and genotoxicity was assessed over 12, 24 and 48h exposure periods. Following 12h, the viability of cells treated with 120 μM-β-sitosterol oxides was reduced to 51·7% relative to control. At 24 and 48 h, both 60 and 120 μM-β-sitosterol oxides caused a significant decrease in cell viability. For comparison, a decrease in viability of cells treated with a cholesterol oxide, 7β-hydroxycholesterol (7β-OH, 30 μM), was evident at 24 h. An increase in apoptotic cells, assessed using Hoechst 33342, indicates that the mode of cell death in U937 cells following exposure to 7β-OH (30 μM) and β-sitosterol oxides (60 and 120 μM) was by apoptosis. The increase in apoptotic cells after 12h following treatment with 120 μM-β-sitosterol oxides was accompanied by a decrease in cellular glutathione. Similarly, 7β-OH (30 μM) treatment resulted in decreased glutathione at 12 h. Catalase activity was not affected by any of the treatments. β-Sitosterol oxides had no genotoxic effects on U937 and V79 cells as assessed by the comet and sister chromatid exchange assays respectively. In general, the results indicate that thermally oxidised derivatives of β-sitosterol demonstrate similar biological effects as 7β-OH in U937 cells, but at higher concentrations.