Clionasterol: a potent inhibitor of complement component C1

Clionasterol (1a), clionasterol monoacetate (1b) and 5alpha,8alpha-epidioxy-24alpha-ethylcholest-6-en-3-ol (2), isolated from the marine sponge Xestospongia exigua, and beta-sitosterol (3) were tested for their influence on the classical (CP) and alternative (AP) pathways of activation of the human complement system in vitro. All the sterols inhibited the CP in a dose-dependent manner but no detectable effect was observed in the AP even at concentrations of 400 microM. Clionasterol was found to be a potent inhibitor of CP (IC50 = 4.1 microM) being ten-fold more active than beta-sitosterol. The presence of the epidioxy group on C-5 and C-8 of compound 2 caused a pronounced decrease of the inhibitory effect. Mechanistic studies on the anticomplementary effect of clionasterol revealed that it interferes with the complement component C1.     

A new phytosterone from Cyanotis arachnoidea

From Cyanotis arachnoidea C.B. Clarke, a new phytosterone named cyanosterone A (1), along with three known compounds--beta-ecdysone (2), ajugasterone C (3) and beta-sitosterol (4) were isolated. The structure of the new compound was determined as 3beta,14alpha,20R,22R-tetrahydroxy-5alpha-cholest-7-en-6-one on the basis of physicochemical properties and spectral analysis.     

露水草的化学成分露水草的化学成分

目的研究露水草的化学成分。 方法应用多种色谱技术进行分离，并结合NMR,MS等波谱解析确定其化学结构。结果分离得到6个化合物，鉴定为20，22-异丙叉基筋骨草甾酮C（1），20，22-异丙叉基β-蜕皮甾酮（2），22-羰基筋骨草甾酮C（3），22-羰基-β-蜕皮甾酮（4），β-谷甾醇（5），胡罗卜苷（6）。结论3为一新化合物，4为一新天然产物。     

Chemoprevention of tumor metastasis by liposomal beta-sitosterol intake

To investigate chemopreventive effect of liposomal beta-sitosterol on tumor metastasis, we prepared liposomal beta-sitosterol composed of egg yolk phosphatidylcholine for oral delivery. Although orally administered beta-sitosterol (4 micromol as beta-sitosterol/mouse) was not absorbed into plasma, the amount of immune response cytokines such as IL-12 and IL-18 was increased in the small intestine after the liposome intake. Moreover, after daily oral administration of the liposome for 7 d, natural killer (NK) cell activity in the mice was increased, suggesting that the immune surveillance activity of mice was enhanced by the liposomal beta-sitosterol intake. Thus, we examined metastatic potential of B16BL6 melanoma cells, which were intravenously injected into mice after sequential administration of liposomal beta-sitosterol for 7 d. The number of metastatic colonies in the lungs was significantly less than that of control group two weeks after the injections of the cells. These results suggest that daily liposomal beta-sitosterol intake prevents tumor metastasis may be due to enhancement of gut immune surveillance systems.     

乌拉尔甘草中新香豆素的化学研究

甘草是一味重要的传统中药,为临床广泛使用。多年来国内外学者对其化学成分的研究颇为重视。近来,用不同溶剂对甘草进行分步提取,以酶和受体生物学指标进行监测,发现乌拉尔甘草(Glycyrrhiza uralensis Fisch)的二氯甲烷提取物对安定受体,胆囊收缩素受体以及二氢还原酶具有明显的抑制作用。为了寻找其中的有效成分,我们对该部分进行了化     

Angiogenic activity of beta-sitosterol in the ischaemia/reperfusion-damaged brain of Mongolian gerbil

Aloe vera continues to be used for wound healing as a folk medicine. We previously reported that A. vera gel has angiogenic activity. In this study, we report upon the isolation of an angiogenic component beta-sitosterol from A. vera and examination of its effect upon damaged blood vessels of the Mongolian gerbil. In a chick embryo chorioallantoic membrane assay, beta-sitosterol was found to have an angiogenic effect. It enhanced new vessel formation in gerbil brains damaged by ischaemia/reperfusion, especially in the cingulated cortex and septal regions, in a dose-dependent fashion (up to 500 microg/kg, p < 0.05, n = 34 - 40). beta-Sitosterol also enhanced the expressions of proteins related to angiogenesis, namely von Willebrand factors, vascular endothelial growth factor (VEGF), VEGF receptor Flk-1, and blood vessel matrix laminin (p < 0.05, n = 6). In addition, the intraperitoneal administration of beta-sitosterol at 500 microg/kg/day for a period of 19 days significantly improved the motion recovery of ischaemia/reperfusion-damaged gerbils as assessed by rota-rod testing (p < 0.001, n = 10). Our results suggest that beta-sitosterol has therapeutic angiogenic effects on damaged blood vessels.     

Influence of 5-tridecylpyrazole-3-carboxylic acid, a new hypolipidaemic agent, on cholesteryl ester formation in rabbit intestinal mucosa

The comparative effects of 5-tridecylpyrazole-3-carboxylic acid (TDPC), beta-sitosterol and melinamide on the esterification of cholesterol (CH) have been investigated in rabbit intestinal microsomes and cytosol in-vitro. The three agents did not show an effect on cholesteryl ester formation by cholesterol esterase (CEase). TDPC and beta-sitosterol did not affect cholesteryl oleate formation from oleoyl CoA by microsomal acyl CoA:cholesterol acyltransferase (ACAT), whereas melinamide significantly inhibited cholesteryl oleate formation. TDPC significantly inhibited the incorporation of oleic acid into cholesteryl oleate, which is associated with acyl CoA synthetase (ACS) plus ACAT in mucosal microsomes, at a concentration of 20-100 microM. On the other hand, 5-tridecylpyrazole-3-carbinol (TDPC-OH) a congener of TDPC, and beta-sitosterol did not show any effect. From these results, it is demonstrated that carboxylic moiety of TDPC is necessary to inhibit ACS in-vitro. According to the kinetic analytical results, it is suggested that TDPC acts as a competitive inhibitor of ACS. These results suggest that the inhibitory effect of TDPC on cholesteryl ester formation may be mediated by an inhibition of ACS activity. It is apparent from the data presented that there are substantial differences between TDPC, beta-sitosterol and melinamide with respect to their action on cholesteryl ester formation in rabbit intestinal mucosa.     

Physical stability of a microcrystalline beta-sitosterol suspension in oil.

Sterols have been shown to reduce plasma cholesterol by blocking the absorption of cholesterol from the gut. The physical properties of crystalline plant sterols limit their use in foods. A coarse-grained structure can be avoided by recrystallisation, a method that affords a reduction in the particle size. A previous work described how to produce a microcrystalline beta-sitosterol suspension. The present study deals with the stability of that suspension. Recrystallisation was carried out by two different methods; one based on rapid the other based on slow cooling, whereby six different compositions were made containing 5-30% of beta-sitosterol and secondly either 5 or 20% water was added. The particle size and habit were evaluated during a 16 weeks storage period (+4 or -19 degrees C) by way of optical microscopy. The crystal structure and degree of crystallinity was analysed by X-ray diffraction. Suspensions can, in most cases, be stored for 16 weeks without any changes to the size and habit. The only evidence of crystal growth came from a suspension with a low sterol concentration at a temperature of +4 degrees C. This is due to the dissolution-diffusion process which is affected by temperature and viscosity. Suspensions containing higher amounts of sterol remained stable, if stored at +4 or -19 degrees C, for 16 weeks. The suspensions included both hemihydrous and monohydrous beta-sitosterol crystals. Suspensions containing less sterol showed greater amounts of monohydrated crystals. This illustrates more water penetration into the crystals. A higher sterol concentration led to a larger number of smaller crystals creating reflections similar to hemihydrated crystals.     

Triterpenes and sterols in the flowers and leaves of Prunus spinosa L. (Rosaceae)

From the flowers of Prunus spinosa L. a mixture of alpha- and beta-amyrine, a mixture of ursolic and oleanolic acids, ursolic acid, a mixture of beta-sitosterol, gamma-sitosterol and stigmasterol and beta-sitosterol 3-O-beta-D-glucopyranoside were isolated. Their structure was determined by spectroscopic methods (GC/MS, IR, 1H and 13C NMR). Chromatographic methods (GC, HPLC, TLC) were employed to determine the presence of the listed compounds in the leaves. Also, the content of beta-sitosterol, ursolic and oleanolic acids in the leaves and flowers of blackthorn was determined by GC/MSD method.     

Beta-sitosterol induces anti-proliferation and apoptosis in human leukemic U937 cells through activation of caspase-3 and induction of Bax/Bcl-2 ratio

Beta-sitosterol is the main dietary phytosterol found in plants and has been shown to inhibit proliferation and induce apoptosis in human solid tumors such as colon and breast cancers. However, the mechanism by which beta-sitosterol induces apoptosis is not completely understood in leukemic cells. This study investigated the mechanism of apoptosis induced by beta-sitosterol in human leukemic U937 cells. beta-Sitosterol induced cytotoxicity and apoptosis in U937 cells in a concentration dependent manner, as measured by hemocytometer counts, fluorescence microscopy, agarose gel electrophoresis, and flow cytometry analysis. The increase in apoptosis induced by beta-sitosterol was associated with down-regulation of Bcl-2, degradation of poly-(ADP-ribose) polymerase (PARP) and phospholipase C (PLC)-gamma1 protein, and activation of caspase-3. beta-Sitosterol induced apoptosis was not associated with changes in the expression of Bcl-xL, Bax, or inhibitor of apoptosis proteins (IAPs). z-DEVD-fmk, a caspase-3 specific inhibitor, blocked caspase-3 activation and PARP degradation, and significantly attenuated beta-sitosterol-induced apoptosis. This suggests that caspase-3 activation is partially essential for beta-sitosterol-induced apoptosis. Bcl-2 overexpression also significantly blocked caspase-3 activation and the decrease in PARP cleavage by beta-sitosterol, and effectively attenuated the apoptotic response to beta-sitosterol. These results show that beta-sitosterol potently induces apoptosis in U937 cells and that beta-sitosterol-induced apoptosis is related to the selective activation of caspase-3 and induction of Bax/Bcl-2 ratio.     

Antilipidemic agents, Part. IV: Synthesis and antilipidemic testing of some heterocyclic derivatives of hexadecyl and cyclohexyl hemisuccinate esters

Two main classes of novel esters containing a hexadecyl or cyclohexyl group and various heterocyclic rings, like quinazolines, triazoles, thiadiazoles and pyrazoles have been synthesized. The first class involves the synthesis of the hexadecyl ester derivatives; namely 3-substituted-2-(hexadecyloxycarbonylpropionylthio)-4(3H)-quinazol inones 4a-c; 4-substituted-3-(4-pyridyl)-5-(hexadecyloxycarbonylpropionylthio)- 4H- 1,2,4-triazoles 6a-c; 5-substituted-2-(hexadecyloxycarbonylpropionylamino)-1,3,4-thiadia zoles 8a-c, 4-[4-(hexadecyloxycarbonyl)phenyl]azo-5-hydroxy-3-methyl-1-phen ylpyrazole 16; and 1-[4-(hexadecyloxycarbonyl) phenyl]-3-methyl-2-pyrazolin-5-one 19. The second class comprises the synthesis of the cyclohexyl ester derivatives; namely 3-substituted-2-(cyclohexyloxycarbonylpropionylthio)-4(3H)-quinazo linones 11a, b, 4-substituted-3-(4-pyridyl)-5-(cyclohexyloxycarbonylpropionylthio) -4 H-1,2,4-triazoles 12a, b and 5-isopropylthio-2-(cyclohexyloxycarbonylpropionylamino)-1,3, 4-thiadiazole 13. The antihypercholesterolemic as well as antihyperlipidemic activities of representative compounds have been studied. All the compounds tested resulted in a decrease in the lipid indices (cholesterol, LDL-cholesterol, HDL-cholesterol and serum triglycerides levels) studied in mice. Compounds 4b, 11a and 12a showed the highest antihyperlipidemic activity; their activities were almost equal to that of beta-sitosterol which was used as a standard.     

Effect of beta-sitosterol concentration and high pressure homogenization on the chlorhexidine release from vesicular gels

Previous studies have confirmed that the phase transition of vesicular gels of hydrogenated phospholipids to the less ordered fluid vesicular state was induced by the increase of the beta-sitosterol ratio in the whole gel system and consequently in the lipid bilayer. The purpose of the present study was to evaluate the influence of the beta-sitosterol portion in the lipid bilayer and the effect of high pressure homogenization on the structural characteristics of the prepared gel systems. In addition the influence of beta-sitosterol on the consequent chlorhexidine release from the obtained vesicles and liposomes was also examined. Lipid mixtures were prepared from different molar ratios of lecithin:sterol components (90:10-65:35 mol%). The obtained mixtures were hydrated with the aqueous solution of chlorhexidine digluconate in order to achieve a 30% (w/w) final concentration of the lipid mixtures and a 4% (w/w) concentration of the drug. One portion of the resultant multilamellar vesicles was homogenized by using high pressure. To characterize the homogenized and non-homogenized systems, transmission electron microscopy of the freeze-fractured samples and differential scanning calorimetry (DSC) were carried out. A vertical type diffusion cell was applied to determine the amount of released chlorhexidine digluconate. Along with the increase in beta-sitosterol concentration, the fluidity of the membrane as well as its permeability also increased. The increased permeability--caused by the higher beta-sitosterol concentration--and the high pressure homogenization, which increased the dispersity and therefore the surface area, enabled a higher amount of chlorhexidine to be released. The increase of drug release was more pronounced in the case of samples prepared with high pressure homogenization.     

Bioactivity-guided isolation of beta-sitosterol and some fatty acids as active compounds in the anxiolytic and sedative effects of Tilia americana var. mexicana

Tilia species have been used as anxiolytics for many years. In a previous study anxiolytic-like effects of a hexane extract of Tilia americana var. mexicana inflorescences were observed in experimental models in mice. To get additional insights into the neuroactive actions of this particular Tilia species, in this study we report a bioactivity guided-fractionation of the extract and separation by column chromatographic methods to isolate three fatty acids and a triterpene identified as beta-sitosterol as major constituents. Our results revealed that the crude extract at 10 and 30 mg/kg I. P. and some pooled fractions at the same dosages potentiated sodium pentobarbital-induced sleeping time and caused a significant increase in the time spent at the open-arm sides in the plus-maze test. A reduction in the exploratory behavioral pattern manifested as ambulatory activity, as well as head dipping and rearing tests was also observed. Further fractionation and purification yielded four major fractions containing fatty acids and beta-sitosterol as the active compounds. A dose-response curve of beta-sitosterol in the range 1 to 30 mg/kg doses indicated that this compound produced an anxiolytic-like action from 1 to 10 mg/kg and a sedative response when the dose was increased to 30 mg/kg, these effects resemble those produced by diazepam (0.1 mg/kg). Our results suggest that hexane extract of Tilia americana var. mexicana produces depressant actions on the central nervous system, at least in part, because of the presence of beta-sitosterol and some fatty acids that remain to be identified.     

Isolation of constituents and anti-complement activity from<Emphasis Type="Italic">Acer okamotoanum</Emphasis>

A novel acylated sterol glucoside (1) along with four known compounds, beta-amyrin acetate (2), 3beta,24-dihydroxytaraxer-14-ene (3), cleomiscosin A (4), and cleomiscosin C (5), were isolated from the leaf and twig of Acer okamotoanum Nakai (Aceraceae). The structure of the new compound was determined to be beta-sitosterol glucoside-3'-O-hexacosanoicate based on chemical and spectroscopic analyses. In addition, the novel compound was found to exhibit a significant inhibitory effect (IC50 value of 0.2 microM) on the complement system activated by the classical pathway.     

Thermoregulatory and locomotor effects of DSIP: paradoxical interaction with d-amphetamine

A low dose (0.1 mg/kg) of peripherally administered delta sleep-inducing peptide (DSIP) caused hypothermia in rats maintained at 4 degrees C but larger doses (1.0 or 3.0 mg/kg) did not. At 22 degrees C all 3 doses of DSIP caused hyperthermia. The interaction of d-amphetamine with DSIP was dependent on ambient temperature; at 4 degrees C DSIP had no effect on d-amphetamine-induced hypothermia, but at 22 degrees C DSIP reversed the usual d-amphetamine-induced effect of hyperthermia to hypothermia. At 4 degrees C, DSIP also potentiated the hypermotility effects of d-amphetamine but blocked it at 22 degrees C. Many rats receiving DSIP (0.1 mg/kg) and d-amphetamine (15.0 mg/kg) at 22 degrees C paradoxically exhibited what appeared to be sleep. This sleep-like effect was not found after several other doses of DSIP and d-amphetamine, chlorpromazine, or at a cold ambient temperature. Sodium methohexital caused apparent sleep in animals treated with DSIP alone or with DSIP and d-amphetamine, but was unable to induce this effect in rats receiving d-amphetamine alone. The results suggest that the effects of peripherally injected DSIP on body temperature are independent from its effects on locomotion and that both effects are dependent upon ambient temperature.     

Repression of acyl-CoA:cholesterol acyltransferase by a traditional herbal medicine (Kampo medicine), Ogi-Keishi-Gomotsu-To-Ka-Kojin.

Ogi-Keishi-Gomotsu-To-Ka-Kojin (OKGK) is a traditional herbal medicine (Kampo medicine) which has been found to ameliorate hypercholesterolemia and hypertriglyceridemia in rats and rabbits. In the present study, the effect of OKGK on acyl-CoA:cholesterol acyltransferase (ACAT) was studied in order to elucidate the mechanism of its antihypercholesterolemic action. Oral administration of OKGK to rats fed a cholesterol-enriched diet for 4 weeks markedly repressed the increase in ACAT activity in the small intestine. In contrast, OKGK did not influence hepatic ACAT activity. These results indicate that OKGK selectively inhibits ACAT activity in the small intestine relative to that in the liver, resulting in a reduction of cholesterol absorption, followed by a decrease in serum cholesterol.     

Modulation of the reactivity of the guinea-pig isolated trachealis by respiratory epithelium: effects of cooling.

1. Examination has been made of the effects of epithelium removal on the reactivity of guinea-pig trachealis to methacholine at 37 degrees C and 22 degrees C, and on responses to activation of the Na+/K(+)-pump by abrupt temperature increase from 22 degrees C to 37 degrees C. 2. At 37 degrees C, epithelium removal increased the sensitivity of isolated tracheal strips to methacholine without affecting the maximum isometric contractile response. Epithelium removal resulted at 22 degrees C in a decrease in sensitivity to methacholine, i.e. an effect opposite to that seen at 37 degrees C. While the maximum response of intact strips to methacholine was enhanced at 22 degrees C, the maximum response of denuded preparations was decreased. 3. The increase in sensitivity to methacholine at 37 degrees C after epithelium removal was mimicked in intact preparations by indomethacin (1 microM). Indomethacin did not mimic the decrease in methacholine sensitivity and maximum response caused by epithelium removal at 22 degrees C. 4. Following incubation at 22 degrees C, abrupt increase in temperature to 37 degrees C elicited relaxation in both epithelium-containing and epithelium-denuded tracheal strips. In epithelium-containing preparations the relaxation was more pronounced and followed by contraction. Ouabain (1 microM) converted the relaxation of denuded preparations to contraction, but was ineffective in intact strips. The relaxation of intact strips was, however, inhibited by a greater ouabain concentration (10 microM). 5. These findings indicate that the modulatory effect of the epithelium is temperature-dependent. In cooled preparations, the epithelium enhances reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)     

蔓性千斤拔化学成分的研究

从豆科植物蔓性千斤拔[Flemingia philippinensis(Merr.et Rolfe)Li]的根中分得六个化合物和一个烷酸的混合物(Ⅰ～Ⅶ)。经理化常数测定和光谱分析证明,化合物Ⅰ和Ⅱ为新化合物,分别命名为蔓性千斤拔素C(Ⅰ)和蔓性千斤拔素D(Ⅱ),其余为已知物,分别鉴定为5,7,3′,4′-四羟基-6,8-双异戊烯基异黄酮(Ⅲ),flemichin D(Ⅳ),n C₂₂～C₃₀烷酸(Ⅴ),β-谷甾醇(Ⅵ)和羽扇豆醇(Ⅶ)。化合物Ⅲ和Ⅳ对P₃₈₈白血病细胞具有显著的抑制作用。     

Two new benzofurans from Gastrodia elata and their DNA topoisomerases I and II inhibitory activities

Two new benzofurans, 1-furan-2-yl-2-(4-hydroxyphenyl)-ethanone (1) and 5-(4-hydroxybenzyloxymethyl)-furan-2-carbaldehyde (2), together with five known compounds, 4-hydroxybenzyl methyl ether (3), 5-(hydroxymethyl)-furfural (4), gastrodin (5), beta-sitosterol (6) and beta-sitosterol glucoside (7) were isolated from the rhizome of Gastrodia elata Blume. In DNA topoisomerase I and II inhibition assays in vitro at a concentration of 20 microM, 1 showed potent inhibitory activity, 66% and 71% inhibition, respectively, compared to the positive control compounds, camptothecin and etoposide, 71% and 22% inhibition, respectively. All of these compounds exhibited weak or no cytotoxicities against human colon carcinoma cell line (HT-29), human breast carcinoma cell line (MCF-7) and human hepatocellular carcinoma cell line (HepG-2).     

Effect of beta-sitosterol on the characteristics of vesicular gels containing chlorhexidine

Previous studies confirm that beta-sitosterol is very effective in altering the molecular packing of soybean lecithin bilayers even more than the cholesterol. The primary aim of the present study was to evaluate the influence of the beta-sitosterol portion in the lipid bilayer on the physical-chemical characteristics of the prepared gel systems, and its influence on the consequent drug release from the liposomes obtained from vesicular phospholipid gels (VPG-s) by redispersion. VPG-s were prepared of different molar ratios of lecithin:sterol components (10:90-35:65 mol%). The mixture was hydrated with the aqueous solution of chlorhexidin digluconate in order to achieve 30% (w/w) final concentration of the lipid mixtures and 4% (w/w) concentration of the drug in each homogenized VPG sample. To characterize the obtained VPG systems optical microscopic examinations using polarized light, differential scanning calorimetry (DSC), photon correlation spectroscopy (PCS), and dynamic surface tension measurements were carried out. Vertical type diffusion cell was applied to determine the amount of released chlorhexidine digluconate. As a result of the surface tension-decreasing effect of beta-sitosterol, the membrane deformability and the dispersity of the system increased. The increased dispersity and fluidity significantly increased the extent of released chlorhexidine from the vesicles.