Osthole (7-methoxy-8[3-methylpent 2-enyl]coumarin), a plant coumarin compound, is extracted from a Chinese herb Cnidium monnieri (L.) CUSS which has been used since ancient times in China as a tonic and aphrodisiac. It was reported that castrated mouse received subcutaneous injections of an alcoholic extract of Cnidium monnieri (L.) CUSS once a day for 21 d, the copulation period appeared, 1) this suggests that this alcoholic extract (including osthole) may have the estrogen-related activities. It has also been found that both total coumarins and osthole, extracted from Cnidium monnieri (L.) CUSS, have the positive effects on bone loss due to ovariectomy in rats, 2,3) where cancellous bone histomorphometric variables in tibiae were preserved. However, even in animal experiments, little is known about the effects of osthole on other skeletal sites or other indices of bone metabolism such as biochemical markers of bone turnover, bone mechanical testing, etc.Ovariectomy induced bone loss in rats and postmenopausal bone loss in humans share many similar characteristics, and similar skeletal response to therapy with 17b-estradiol. 4,5) These similarities are strong evidence that the ovariectomized (OVX) rat bone loss model is suitable for studying the prevention and treatment of postmenopausal bone loss. Furthermore, the femoral neck of OVX rats, is a more clinically relevant sample site than other skeletal sites (i.e., proximal tibia) for preclinical testing of new therapeutic agents for the prevention and treatment of osteopenia. [6][7][8] The purpose of this study was to examine whether osthole has positive effects on bone loss due to OVX using the femoral neck of OVX rats; and, if so, whether osthole functions at the tissue level in a manner similar to 17b-estradiol. MATERIALS AND METHODSExperimental Protocols Twenty-four 3-month-old virgin female Wistar rats (220Ϯ12 g, Charles River Inc. Tokyo, Japan) were used for the experiment. The animals were kept for 5 d before the onset of the experiment to acclimatize to our laboratory conditions (the room temperature was 25°C with a 12 h/12 h light/dark cycle), then 18 rats were OVX and 6 rats were sham-operated under anesthesia with intraperitoneal (IP) injection of sodium pentobarbital at a dose of 30 mg/kg body weight. After operation, rats were kept in separate cages and fed standard diet before being sacrificed. To prevent hyperphagia associated with ovariectomy, the OVX rats were pair-fed to the mean intake of those in the sham group.9) All rats were allowed free access to drinking ion-exchanged distilled water for the duration of the whole experiment.All rats were untreated for 2 weeks after surgery and divided into four groups (6 rats per group). The first group was sham-operated upon and received solvent vehicle (97% corn oil and 3% ethanol, 1.0 ml/kg). Groups 2 to 4 were OVX. Group 2 received solvent vehicle (same the sham rats), Group 3 received 17b-estradiol (Sigma Chemical Company, St. Louis, MO, U.S.A.) 30 mg/kg and Group 4 received osthole (Wako Pure Chem...

Linoleate (C18: 2) and oleate (C18: 1), but not stearate (C18: 0), induced tail removal in cercariae. Linoleate stimulated tail loss more strongly than oleate did. Tail loss induced by linoleate was significantly suppressed by incubating cercariae with ethyleneglycol-bis-(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA). Preincubation of cercariae with EGTA for 5 min caused further inhibition of the tail loss. Calcium ionophore A23187 (A23187) increased the cercarial tail-loss rate. When A23187 was combined with linoleate at 0.03 mM, an additive effect on tail loss appeared, whereas the ionophore in combination with linoleate at 0.3 mM had no such effect. EGTA almost completely abolished cercarial tail loss induced by linoleate at both 0.03 and 0.3 mM in the presence and absence of A23187. Linoleate at 3 mM provoked cercarial tail loss even in the presence of EGTA, although the effect of oleate at 3 mM disappeared. Under these conditions, the effect of linoleate was synergistically enhanced by the combination with A23187. A similar, but not significant, synergism took place in cercariae stimulated by oleate. These findings suggest that unsaturated fatty acids enhance calcium influx into cercariae, resulting in triggering tail loss, and, furthermore, that the fatty acids have other potentiating effects on cercarial tail loss. Protein kinases play an insignificant role in fatty acid-induced cercarial tail loss, because a protein kinase C inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), and an inhibitor of various protein kinases, staurosporine, had little or no effect on cercarial tail loss induced by linoleate at 3 mM.

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SUMMARYIt has been indicated that the anti-estrogen agent, tamoxifen, developed for the treatment of breast cancer, may act on the vascular system as an estrogen agonist. However, to our knowledge few reports suggest that tamoxifen exerts anti-atherogenic actions. In the present study, we evaluated the anti-atherosclerotic effects of tamoxifen in ovariectomized cholesterol-fed rabbits. Ovariectomized rabbits were fed a 1% cholesterol diet and divided into 4 groups: control group (C, n=5); estrogen treatment (E, n=6); low-dose tamoxifen treatment (0.5 mg/kg) (LT, n=6); and high-dose tamoxifen (1.0 mg/kg) (HT, n=7). After 6 weeks, both Oil red O-positive areas on the intimal surfaces of aortae and the ratios of intimal to medial areas (I/M ratios) measured from cross-sections of aortae were significantly lower in groups E, LT and HT compared with group C. Although there were no significant differences in serum NOx (NO 2 and NO 3 ) levels among the 4 groups, NOx levels were slightly higher in groups E, LT and HT than group C. Acetylcholine (ACh) was administered to all animals, and the responses of ear arteriole diameters were compared among the 4 groups. While ear arteriole diameters were significantly decreased in group C, no significant changes were observed in groups E, LT or HT following ACh administration. Ratios of ear arteriole diameters after to before ACh administration were significantly greater in groups E, LT and HT compared to group C. These findings suggest that tamoxifen exerts anti-atherosclerotic effects, and that these effects are attributed to the maintenance of vascular endothelial function. (Jpn Heart J 2002; 43: 545-558) Key words: Tamoxifen, Estrogen, Atherosclerosis, Vascular endothelial function, NO WHILE the incidence of cardiovascular disease in premenopausal women is less than half that in men of similar age, its rate of occurrence markedly increases following menopause, with the coronary heart disease incidence in women over the age of 75 approaching the rates of men. 1) In vitro studies demonstrate that estrogen exerts anti-atherogenic actions by inducing LDL receptor activity, 2)From the

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