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The methanolic extract from fresh stems of Cistanche tubulosa (Orobanchaceae) was found to show hepatoprotective effects against D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced liver injury in mice. From the extract, three new phenylethanoid oligoglycosides, kankanosides H-1 (1), H-2 (2), and I (3), were isolated together with 16 phenylethanoid glycosides (4-19) and two acylated oligosugars (20, 21). The structures of 1-3 were determined on the basis of spectroscopic properties as well as of chemical evidence. Among the isolates, echinacoside (4, IC50 = 10.2 mu M), acteoside (5, 4.6 mu M), isoacteoside (6, 5.3 mu M), 2’-acetylacteoside (8, 4.8 mu M), and tubuloside A (10, 8.6 mu M) inhibited D-GalN-induced death of hepatocytes. These five isolates, 4 (31.1 mu M), 5 (17.8 mu M), 6 (22.7 mu M), 8 (25.7 mu M), and 10 (23.2 mu M), and cistantubuloside B-1 (11, 21.4 mu M) also reduced TNF-alpha-induced cytotoxicity in L929 cells. Moreover, principal constituents (4-6) exhibited in vivo hepatoprotective effects at doses of 25-100 mg/kg, po. (C) 2010 Elsevier Ltd. All rights reserved.