Antimicrobial constituents from the stem bark of Feronia limonia.

The stem bark of Feronia limonia (Fam. Rutaceae) yielded (-)-(2S)-5,3'-dihydroxy-4'-methoxy-6",6"-dimethylchromeno-(7,8,2",3")-flavanone along with several known compounds including an alkaloid, five coumarins, a flavanone, a lignan, three sterols and a triterpene. The structures of these compounds were determined by spectroscopic methods, mainly 1D and 2D NMR. The antimicrobial screening of compounds by a microdilution technique resulted in MICs in the range 25-100 microg/ml. Other biological activities of the known compounds are also discussed.     

Mechanism of action of antiatherogenic and related effects of Ficus bengalensis Linn. flavonoids in experimental animals

One month treatment of alloxan diabetic dogs with a glycoside, viz. leucopelargonin derivative (100 mg/kg/day) isolated from the bark of F. bengalensis decreased fasting blood sugar and glycosylated haemoglobin by 34% and 28% respectively. Body weight was maintained in both the treated groups while the same was decreased significantly by 10% in the control group. In cholesterol diet fed rats, as the atherogenic index and the hepatic bile acid level and the faecal excretion of bile acids and neutral sterols increased, the HMGCoA reductase and lipogenic enzyme activities in liver and lipoprotien lipase activity in heart and adipose tissue and plasma LCAT activity and the incorporation of labelled acetate into free and ester cholesterol in liver decreased significantly. On treatment with the two ficus flavonoids, viz. leucopelargonin and leucocyanin derivatives and another flavonoid quercetin (100 mg/kg/day) the above said effects except on bile acids and sterols and lipogenic enzymes were significantly reversed in the cholesterol fed rats. However in the treated rats the hepatic level of bile acids and the faecal excretion of bile acids and neutral sterols still further increased and the action of lipogenic enzyme glucose 6 phosphate dehydrogenase was still further decreased. These effects of leucopelargonidin and quercetin were better than that of the second. Toxicity studies are required to be carried out to find out if the ficus flavonoids could be used as health promoters as they are hypocholesterolemic and antioxidant in action.     

Chemical and Biophysical Changes in the Plasma Membrane during Cold Acclimation of Mulberry Bark Cells (Morus bombycis Koidz. cv Goroji)

The lipid and protein composition of the plasma membrane isolated from mulberry (Morus bombycis Koidz.) bark cells was analyzed throughout the cold acclimation period under natural and controlled environment conditions. There was a significant increase in phospholipids and unsaturation of their fatty acids during cold acclimation. The ratio of sterols to phospholipids decreased with hardiness, primarily due to the large increase in phospholipids. The fluidity of the plasma membrane, as determined by fluorescent polarization technique, increased with hardiness. Electrophoresis of plasma membrane proteins including glycoproteins revealed change in banding pattern during the early fall to winter period. Some of the protein changes could be related to growth cessation and defoliation. However, minor changes in proteins also occurred during the most active period of hardening. Changes in glycoproteins were coincident both with changes in growth stages and with the development of cold hardiness.     

Constituents of Acacia cedilloi and Acacia gaumeri. Revised structure and complete NMR assignments of resinone

The rare lupene derivative named resinone has only been isolated before from Fluorensia resinosa. We now report the isolation of this compound from the bark of the new recently described Acacia cedilloi (Fabaceae), and the revision of its structure to 16beta-hydroxylup-20(29)-en-3-one, based on NMR and MS spectral data. The detailed 1H and 13C NMR assignments of resinone and its acetate achieved by 1D and 2D NMR experiments (including DEPT, COSY, HMQC and HMBC) are reported. In addition, the study of A. cedilloi and A. gaumeri afforded the known related lupenes lupeol and lupenone, the acyclic squalene, the sterols beta-sitosterol, stigmasta-7,22-dien-3beta-ol (spinasterol) and stigmasta-5,22,25-trien-3beta-ol (22-dehydroclerosterol) as well as alpha-tocopherol and beta-carotene.     

The Chemical Constituents of Amoora yunnanensis

Two new sterols, 3β,7α,16β-trihydroxy-stigmast-5,22-diene (1), 3β,7α,16β-trihydroxy-stigmast-5-ene (2), were isolated together with six known compounds, ergosta-5,24(28)-dien-3β,7α-diol (3), ergosta-5,24(28)-dien-3β,7β,16β-triol (4), β-amyrone (5), β-amyrin (6), 11α,12α-epoxy-14-taraxeren-3-one (7), and 6-guaiene-4α,10α-diol (8) from the EtOH extract of the bark of Amoora yunnanensis (H. L. Li) C. Y. Wu. Their structures were deduced on the basis of spectral data.     

Terpenoid and other extractives of western white pine bark

A detailed chemical analysis of the benzene extract of western white pine bark was conducted. The extract consisted of 13% phlobaphenes, 18% strong acids, 21% polar weak acids, 6.5% fatty acids, 9.5% resin acids, and 32% neutrals. The fatty acids consisted mainly of C_20:0, C_22:0, and C_24:0 acids. The resin acids were identified as: isopimaric, anticopalic, dehydroabietic, sandaracopimaric, abietic, 6,8,11,13-abietatetraen-18-oic and pimaric acids. The neutrals on saponification gave fatty acids, sterols, wax alcohols, nonsaponifiables, and other components. The esterified fatty acids consisted primarily of the C_16:0, C_18:0, C_20:0 and C_24:0 acids. The sterols included major amounts of sitosterol, campesterol, and stigmasterol, and traces of cholesterol. Over 70 individual compounds were isolated and identified from the nonsaponifiables. These included borneol, sesquiterpenes, diterpenes, steroidal ketones, as well as lanostane and serratane triterpenes. The characterization of12 new natural products or natural products isolated for the first time from Pinus species is reported.     

The incorporation of mevalonate-[2-14C] into the sterol fractions of Calendula officinalis

The incorporation of mevalonate-[2-¹⁴C] into the free sterols, steryl esters, steryl glucosides, acylated steryl glucosides and water-soluble complexes was investigated and the sterols of each fraction were separated into stanols, Δ⁷ sterols, Δ⁵ sterols, stigmasterol, clerosterol and methylene-cholesterol. The stanols and Δ⁷ sterols were more strongly labelled in the steryl esters than in the free sterols. The Δ⁵ sterols and stigmasterol were more intensively labelled in the free sterols than in the steryl esters. All sterol types were more labelled in the steryl glycosides than in the acylated steryl glucosides. Stanols were probably formed from Δ⁷ or Δ⁵ precursors.     

Supplementation with sterols improves food quality of a ciliate for Daphnia magna

Experimental results provide evidence that trophic interactions between ciliates and Daphnia are constrained by the comparatively low food quality of ciliates. The dietary sterol content is a crucial factor in determining food quality for Daphnia. Ciliates, however, presumably do not synthesize sterols de novo. We hypothesized that ciliates are nutritionally inadequate because of their lack of sterols and tested this hypothesis in growth experiments with Daphnia magna and the ciliate Colpidium campylum. The lipid content of the ciliate was altered by allowing them to feed on fluorescently labeled albumin beads supplemented with different sterols. Ciliates that preyed upon a sterol-free diet (bacteria) did not contain any sterols, and growth of D. magna on these ciliates was poor. Supplementation of the ciliates' food source with different sterols led to the incorporation of the supplemented sterols into the ciliates' cells and to enhanced somatic growth of D. magna. Sterol limitation was thereby identified as the major constraint of ciliate food quality for Daphnia. Furthermore, by supplementation of sterols unsuitable for supporting Daphnia growth, we provide evidence that ciliates as intermediary grazers biochemically upgrade unsuitable dietary sterols to sterols appropriate to meet the physiological demands of Daphnia.     

Yeast-like symbiotes as a sterol source in anobiid beetles (Coleoptera,Anobiidae): possible metabolic pathways from fungal sterols to 7-dehydrocholesterol

Insects are unable to synthesize sterols and require exogenous sterol sources for their normal development and reproduction. A few exceptions are insects associated with symbiotic yeasts or fungi. We analyzed sterols by GC-MS in two anobiid beetles (Lasioderma serricorne and Stegobium paniceum), their intracellular yeast-like symbiotes (YLS), and their diets in order to clarify the sterols synthesized by YLS and the metabolic pathways of the sterols in the beetles. Several C(27), C2(8), and C(29) saturated and unsaturated sterols were identified; the predominant sterols were cholesterol and 7-dehydrocholesterol in the anobiid beetles and ergosterol in the YLS. Most sterols detected in YLS were those known in the late pathway of the ergosterol biosynthesis in yeasts and most of the sterols in the beetles appear to be intermediate metabolites from YLS sterols to 7-dehydrocholesterol. The anobiid beetles appear to use ergosterol and 5-dihydroergosterol as sources for 7-dehydrocholesterol.     

Quantitative aspects of free and esterified sterols in Saccharomyces cerevisiae under various conditions.

For extraction of free and esterified sterols from yeast cells, a method was devised in which both forms of sterols were extracted with light petroleum after the treatment of the cells with acetone, and then with dimethylsulfoxide. The content of sterol esters in the cells under aerobic conditions markedly increased with time, amounting to 95% of the total sterols under some conditions. However, the formed sterol esters were decreased, accompanied with an increase of free sterols, when the cells were put under anaerobic conditions. Variations of radioactivities of both sterols which had been labeled in the side chain by incubation of the cells with [Me[-14C]methionine were examined on the cells grown under various conditions. No variation was observed on the cells under aerobic conditions. On the other hand, the labeled esters were hydrolyzed to yield free sterols in the cells under anaerobic conditions. In the cells under aerobic conditions, the free sterols were found to consist mainly of ergosterol, whereas the esterified sterols contained considerable amounts of zymosterol, lanosterol, and other intermediate sterols besides ergosterol.     

Plasma membrane rafts complete cholesterol synthesis by participating in retrograde movement of precursor sterols

Mammalian cells synthesize significant amounts of precursor sterols, in addition to cholesterol, at the endoplasmic reticulum (ER). The newly synthesized sterols rapidly move to the plasma membrane (PM). The mechanism by which precursor sterols move back to the ER for their enzymatic processing to cholesterol is essentially unknown. Here we performed pulse-chase experiments and showed that the C29/C30 sterols rapidly move from the PM to the ER and are converted to cholesterol. The retrograde precursor sterol transport is largely independent of the Niemann-Pick type C proteins, which play important roles in late endosomal cholesterol transport. In contrast, disrupting lipid rafts significantly retards the conversion of C29/C30 and C28 sterols to cholesterol, causing the accumulation of precursor sterols at the PM. Our results reveal a previously undisclosed function of the PM lipid rafts: they bring cholesterol biosynthesis to completion by participating in the retrograde movement of precursor sterols back to the ER.     

Inhibition of Sterol Biosynthesis in Monilinia fructigena by the Fungicide,S-1358

When Moniiinia fructigena was treated with S–1358 at a concentration of 10 μm, both quality and quantity of digitonin-precipitable sterols were markedly altered. The amount of ergosterol which is a major sterol in the control culture was reduced by S–1358 and the concomitant accumulation of obtusifoliol (one of 4α-methyl sterols) and 24-methylenedihy-drolanosterol (one of 4,4-dimethyl sterols) was observed. The time course study of acetate-U-¹⁴C incorporation into the digitonin-precipitable sterols revealed that 4α-methyl sterols accumulated slowly in the treated culture, while 4,4-dimethyl sterols accumulated rapidly. The accumulation of the sterols containing “extra1” methyl groups suggests that S–1358 blocks demethylation reactions in the conversion from lanosterol to ergosterol in M. fructigena.     

Sterols of organs involved in brood food production and of royal jelly in honey bees

The sterols of the organs (hypopharyngeal and mandibular glands and honey stomachs) involved in worker and queen honey bee brood food production, royal jelly and intact nurse bees were analyzed to obtain information on the selective transfer of specific sterols from one generation to the next. No appreciable increase in the percentage of 24-methylenecholesterol, relative to the total sterols isolated from intact honey bee (Apis mellifera L.), prepupae or adults, was found in the hypopharyngeal or mandibular glands or the honey stomachs from nurse bees reared in colonies fed a chemically-defined diet supplemented with 24-methylenecholesterol. The sterols of these organs contained higher levels of cholesterol than did the sterols of whole body extracts. The other major sterols, sitosterol and isofucosterol, occurred at relative concentrations comparable to whole body extracts. Also, there were higher levels of cholesterol in the sterols from glandular tissues of nurse bees maintained on pollen and sucrose solution than in sterols isolated from intact insects. In a separate study, royal jelly collected over a 6-day period had much higher relative percentages of 24-methylenecholesterol and lower levels of sitosterol and isofucosterol than did the pollen fed to these colonies. The sterols of nurse bees in the latter study had an intermediate concentration of 24-methylenecholesterol. The significance of these findings relative to the unique selective transfer of specific sterols from the diet or from endogenous sterol pools of the nurse bees from generation to generation in the honey bee is discussed.     

Phytosterol structure and its impact on feeding behaviour in the generalist grasshopper Schistocerca americana

Abstract.Sixth-stadium nymphs of the grasshopper Schistocerca americana (Drury) (Orthoptera: Acrididae) were observed in a series of experiments designed to measure feeding behaviour in response to suitable and unsuitable phytosterols. In the first experiment, grasshoppers were presented with artificial diet that contained either sitosterol, a suitable phytosterol, or a spinach lipid extract which contained only unsuitable sterols as well as other spinach lipids. The diet with the spinach lipid extract, but not the sitosterol diet, evoked a deterrent response. To determine if the spinach sterols were responsible for the deterrent response, a second experiment was performed where the spinach lipid extract was separated into three lipid classes, including desmethyl sterols, dimethyl sterols and the remaining spinach lipids. Grasshoppers presented with artificial diet containing the desmethyl sterols (the end-product sterols in spinach) exhibited deterrent responses. Finally, feeding behaviour to a suite of different sterols, including cholesterol (suitable), stigmasterol (unsuitable), and lathosterol (unsuitable), was observed; these sterols were selected because they show variation in the position of double bonds. Grasshoppers presented with diets containing unsuitable sterols again exhibited deterrent responses. Overall, the deterrent effect was strongest when sterols with a double bond at position 22 were in the diet.     

The effect of sterol structure on membrane lipid domains reveals how cholesterol can induce lipid domain formation

Detergent-insoluble membrane domains, enriched in saturated lipids and cholesterol, have been implicated in numerous biological functions. To understand how cholesterol promotes domain formation, the effect of various sterols and sterol derivatives on domain formation in mixtures of the saturated lipid dipalmitoylphosphatidylcholine (DPPC) and a fluorescence quenching analogue of an unsaturated lipid was compared. Quenching measurements demonstrated that several sterols (cholesterol, dihydrocholesterol, epicholesterol, and 25-hydroxycholesterol) promote formation of DPPC-enriched domains. Other sterols and sterol derivatives had little effect on domain formation (cholestane and lanosterol) or, surprisingly, strongly inhibit it (coprostanol, androstenol, cholesterol sulfate, and 4-cholestenone). The effect of sterols on domain formation was closely correlated with their effects on DPPC insolubility. Those sterols that promoted domain formation increased DPPC insolubility, whereas those sterols that inhibit domain formation decreased DPPC insolubility. The effects of sterols on the fluorescence polarization of diphenylhexatriene incorporated into DPPC-containing vesicles were also correlated with sterol structure. These experiments indicate that the effect of sterol on the ability of saturated lipids to form a tightly packed (i.e., tight in the sense that the lipids are closely packed with one another) and ordered state is the key to their effect on domain formation. Those sterols that promote tight packing of saturated lipids promote domain formation, while those sterols that inhibited tight packing of saturated lipids inhibited domain formation. The ability of some sterols to inhibit domain formation (i.e., act as "anti-cholesterols") should be a valuable tool for examining domain formation and properties in cells.     

Minor and trace sterols in marine invertebrates. 61. Isolation and structure elucidation of new A-nor sterols from the marine sponge Phakellia aruensis

An examination of the Australian sponge Phakellia aruensis led to the isolation and identification of sterols with six different nuclei. Eight new sterols were isolated which included three delta 15-A-nor sterols, three delta 7-A-nor sterols, and two saturated A-nor sterols. Their structures were established through mass spectrometry and 1H-NMR spectral studies.     

Schistosoma haematobium: neutral lipid composition and release by adults maintained in vitro

Thin-layer chromatographic analyses showed that the major neutral lipid fractions of whole-worm extracts of male and female adult Schistosoma haematobium were free sterols, triacylglycerols and sterol esters. Worm-free incubates of adult worm-pairs contained free sterols only. The major fractions of worm-free incubates from separated worms were free fatty acids and free sterols; traces of triacylglycerols and sterol esters were also detected. Females incubated in a group of ten released more free fatty acids than ten incubated singly. Males incubated singly released more free sterols than a similar number incubated in a group. Females released more free sterols than males.     

Heritability of plasma noncholesterol sterols and relationship to DNA sequence polymorphism in ABCG5 and ABCG8

The plasma concentrations of cholesterol precursor sterols and plant sterols vary over a 5- to 10-fold range among normolipidemic individuals, and provide indices of the relative rates of cholesterol synthesis and fractional absorption. In the present study, we examined the relative contributions of genetic and environmental factors to variation in the plasma concentrations and sterol-cholesterol ratios of five noncholesterol sterols, including the 5alpha-saturated derivative of cholesterol (cholestanol), two precursors in the cholesterol biosynthesis pathway (desmosterol and lathosterol), and two phytosterols (campesterol and sitosterol). Plasma sterol concentrations were highly stable in 30 individuals measured over a 48 week period. Regression of offspring sterol levels on the parental values indicated that plasma levels of all five noncholesterol sterols were highly heritable. Analysis of monozygotic and dizygotic twin pairs also indicated strong heritability of all five sterols. Two common sequence variations (D19H and T400K) in ABCG8, an ABC half-transporter defective in sitosterolemia, were associated with lower concentrations of plant sterols in parents, and in their offspring.Taken together, these findings indicate that variation in the plasma concentrations of noncholesterol sterols is highly heritable, and that polymorphism in ABCG8 contributes to genetic variation in the plasma concentrations of plant sterols.     

Sterol content of the Myxomycetes Physarum polycephalum and P. flavicomum.

The sterol content of two Myxomycetes, Physarum polycephalum and P. flavicomum has been examined. The sterols of the two species are apparently identical, the two major sterols in each being poriferasterol and 22-dihydroporiferasterol. Threee minor sterols are probably delta5-ergostenol, ergostanol, and poriferastanol. The triterpenoids of the two species differ in that, though lanosterol was identified in both, 22-dihydrolanosterol was indicated only in P. flavicomum. The occurrence of lanosterol together with a typical mixture of plant sterols is somewhat unusual.     

A simplified micro-method for quantification of fecal excretion of neutral and acidic sterols for outpatient studies in humans.

A simple and precise micro-method for measurement of daily fecal excretion of neutral and acidic sterols has been developed which utilizes sitostanol (24-ethyl-5 alpha-cholestane-3 beta-ol) as fecal flow and recovery marker. Extractions of sterols were performed from 50 microliters of fecal homogenate (feces-water 1:1), and analyses of neutral and acidic sterols were carried out by gas-liquid chromatography. The method is sensitive, precise, and easy to perform; the intra-assay variability yielded coefficients of variations of 1.9% and 3.5% (n = 6) for neutral and acidic sterols, respectively. The results from this method were compared with those obtained with the standard fecal flow marker chromic oxide. The correlation coefficients between the two markers were compared in 16 subjects and were 0.938 and 0.998 for excretion of neutral sterols and acidic sterols, respectively. Comparison of the fecal excretion of neutral and acidic sterols in 12 subjects determined from frozen samples and aliquots (approximately 1 g) sent by ordinary mail to the laboratory (transport time 1 to 5 days) gave identical results using sitostanol as fecal flow marker (818 +/- (SEM) 85 mg/day vs. 838 +/- 89 mg/day for neutral sterols and 417 +/- 59 mg/day vs. 414 +/- 60 mg/day for acidic sterols). The new micro-method is ideally suited for research laboratories in need of a simple, accurate, inexpensive, and high through-put method for measuring daily fecal excretion of neutral and acidic sterols, as well as total cholesterol synthesis, and can be performed on an outpatient basis.