Antileishmanial activity of indole alkaloids from Aspidosperma ramiflorum

The present study was designated to evaluate the antileishmanial activity of acid and basic fractions that were obtained after acid-basic extraction, from ethanolic 70% crude extract and pure compounds from the stem bark of Aspidosperma ramiflorum. The basic alkaloidal fraction presented a good activity against the extracellular form (promastigotes) of Leishmania (L.) amazonensis (LD(50) value<47 microg/ml). Based on these findings, the basic fraction was fractionated on silica gel column chromatography in a bioassay-guided fractionation affording individual purified ramiflorines A and B. Both ramiflorines A and B showed significant activity against Leishmania (L.) amazonensis (LD(50) values of 16.3+/-1.6 microg/ml and 4.9+/-0.9 microg/ml, respectively). Our results are promising, showing that these compounds are biologically active against Gram-positive bacteria.     

The insulin sensitizing effect of homoisoflavone-enriched fraction in Liriope platyphylla Wang et Tang via PI3-kinase pathway

In the present study, we screened candidates for enhancing insulin action, using glucose uptake as an indicator, from Liriope platyphylla Wang et Tang (LPWT) extract, Liliaceae, in 3T3-L1 adipocytes. The mechanism of insulin sensitizing action in the fractions was also investigated. LPWT extract with 70% MeOH was sequentially separated with Diaion HP-20 and silica gel column chromatography. The 9:1 fraction from silica gel column chromatography increased glucose uptake with 1 ng/mL up to glucose uptake with 50 ng/mL insulin. The 9:1 fraction, determined as homoisoflavone-enriched fraction, worked as an insulin sensitizer. It increased insulin stimulated glucose uptake in 3T3-L1 adipocytes, insulin responsive cells, through increased glucose transporter 4 (GLUT4) contents in the plasma membrane. GLUT4 translocation was increased through insulin receptor substrate 1 (IRS1)-PI3 kinase-Akt signaling mechanism. Thus, homoisoflavone-enriched fraction in LPWT extract played an important role as an insulin sensitizer in adipocytes.     

Diterpenoids from the fruits of Vitex trifolia

An abietane-type diterpene, named vitetrifolin A, and two labdane-type diterpenes, named vitetrifolins B and C, were isolated from the acetone extract of the fruits of Vitex trifolia L. (Viticis Fructus; Verbenaceae) along with three known diterpenes, rotundifuran, dihydrosolidagenone and abietatriene 3beta-ol. The structures of these compounds were elucidated on the basis of spectroscopic analysis, X-ray crystallographic analysis and chemical evidence.     

Chemical cues from Spodoptera litura larvae elicit prey-locating behavior by the predatory stink bug, Eocanthecona furcellata

Two chemical compounds eliciting prey-locating behavior of the predatory stink bug, Eocanthecona furcellata, were isolated from solvent extracts of Spodoptera litura larvae and identified. A hexane eluted fraction from silica gel chromatography of the solvent extracts of S. litura larvae was attractive to E. furcellata nymphs when assayed with a linear track olfactometer. The hexane fraction was found to contain n-pentadecane (2500 ng/larva), n-tetracosane (54 ng/larva), n-heptadecane (41 ng/larva), n-heptacosane (61 ng/larva), n-nonacosane (147 ng/larva), n-hentriacontane (200 ng/larva) and squalene (323 ng/larva). The synthetic n-pentadecane was attractive to the bugs, although its activity was slightly lower than that of the hexane fraction. A mixture of synthetic n-pentadecane and the other six hydrocarbons was also attractive, although no improved attractiveness was observed by the addition of the latter compounds. On the other hand, a 15%-ether-in- hexane eluted fraction from silica gel chromatography stimulated bugs to display a proboscis-protruding behavior. A neutral-layer fraction of the 15% fraction, which contained E-phytol (480 ng/larva), also elicited this behavior. Synthetic E-phytol had the same effect on the predators as the neutral-layer fraction.     

Substructures and Polypeptides of Visna Virus

The protein of Visna virus, disrupted by 8 M guanidine hydrochloride and heating, was resolved into 10 polypeptides by agarose gel column chromatography in 6 M guanidine hydrochloride. Two of the peaks contained glycopolypeptides. Nonidet-disrupted virions were resolved into two fractions by potassium tartrate gradient centrifugation, with densities of 1.08 and 1.24 g/ml, respectively. About 70% of the viral DNA polymerase directed by added template was released into the light fraction, in which very little endogenous enzyme activity was detected. Also released into the light fraction were all of the glycopolypeptides, 50% of the viral RNA, and a part of each of the other viral protein components. The data indicate that extensive degradation of subviral structures occurred, even under mild conditions for virion disruption. The 1.24-g/ml fraction was composed of 50% of the viral RNA, most of the endogenous DNA polymerase activity (80%), and a major internal polypeptide (GuHCl6) with an estimated mol wt of 28,000. Two other polypeptides were also consistently detected in the heavy fraction, but they constituted less than 25% of the ribonucleoprotein complex, compared with 75% for GuHCl6.     

Susceptibility of some clinical isolates of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> to fractions from the aerial parts of <Emphasis Type="Italic">Leuzea carthamoides</Emphasis>

The antimicrobial activity of the dichloromethane extract from aerial parts of Leuzea carthamoides DC. was tested in vitro against 19 Staphylococcus aureus strains (ATCC 25923, CNCTC Mau 43/60, clinical isolates). The extract was fractionated by column chromatography on silica gel into six fractions (petroleum ether, toluene, dichloromethane, ethyl acetate, methanol and water). The minimum inhibitory concentrations (MICs) of the fractions ranged from 64 to 1024 μg/mL. An ethyl acetate fraction (EA 1) with the widest range of activity inhibited all of the strains with MIC in the range 128–512 μg/mL. This fraction exhibited potent activity against strains which showed associated resistance to oxacillin, ciprofloxacin and erythromycin.     

In vitro antibacterial activity of a 7-O-beta-D-glucopyranosyl-nutanocoumarin from Chaptalia nutans (Asteraceae)

Ethanolic crude extracts from the roots of Chaptalia nutans, traditionally used in Brazilian folk medicine, were screened against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa by using the disk diffusion test technique. S. aureus with 14 mm inhibition zone was considered susceptible. E. coli and P. aeruginosa without such a zone were considered resistant. As a result of this finding, the ethanolic crude extract was fractionated on silica gel column chromatography into five fractions. The ethyl acetate fraction was active against S. aureus and Bacillus subtilis. Further column chromatography separation of the ethyl acetate fraction afforded 30 fractions, which were assayed against S. aureus. Fractions 16 and 17 showed inhibition zones with S. aureus, indicating the presence of active compounds, and were subjected to purification by repeated preparative thin layer chromatography. The pure compound 7-O-beta-D-glucopyranosyl-nutanocoumarin inhibited B. subtilis and S. aureus at concentrations of 62.5 g/ml and 125 g/ml, respectively. The antibacterial property of C. nutans appears to have justified its use for the treatment of wounds, which are contaminated through bacterial infections.     

Trace amounts of ganglioside GM1 in human milk inhibit enterotoxins from Vibrio cholerae and Escherichia coli

Gangliosides were isolated from human milk fat and purified by silica gel column chromatography and high performance liquid chromatography (HPLC). Low amounts of the ganglioside GM1, detected by high performance thin layer chromatography (HPTLC)-immunoassay, were found in all fractions with enterotoxin-inhibitory activity, while fractions without GM1 were inactive. It is concluded that GM1 is responsible for enterotoxin-inhibitory activity in the ganglioside fraction from human milk.     

Identification and quantitation of 16α-hydroxy c21 steroid sulphates in plasma from pregnant women

Plasma steroids from 11 women in the last trimester of pregnancy were separated into conjugate class by chromatography on Sephadex LH-20. Fractions corresponding to mono- and disulphate conjugates were solvolyzed and the free steroids were separated by lipophilic gel chromatography into groups containing mono-, di- and trihydroxy steroids, respectively. Trimethylsilyl ether derivatives were prepared and repetitive-scanning gas chromatography-mass spectrometry (GC-MS) was used to characterize and quantitate the following 16α-hydroxy steroids in the monosulphate fraction: 3α,16α:-dihydroxy-5α-pregnan-20-one (range 8–80 ng/ml), 3β,16α-dihydroxy-5α-pregnan-20-one (6–55 ng/ml), 5α-pregnane-3α,16α,20α-triol (9–31 ng/ml) and 5α-pregnane-3β,16α,20α-triol (11–77 ng/ml). These compounds were also present as disulphates although at 5–50 times lower concentrations.     

The binding of silica to proteins from plasma and lungs of rat: in vitro

Silica dissolving out from the slate dust was found to bind with plasma protein and purified bovine serum albumin. At 24 h of incubation at 37 degrees C binding affinity of silica (microgram of silica bound/mg of protein) with plasma protein and bovine serum albumin was found to be 0.59 and 0.44, respectively. By molecular exclusion chromatography using Sephadex G-200, silica binding protein of plasma was determined to be of mol. wt. around 67000. Similar proteins having silica binding capacity (mol. wt. 70000 and 85000) were also found in rat lung but these proteins unlike their plasma counterpart were glycoprotein in nature. Polyacrylamide gel electrophoresis of plasma and protein rich lung fraction show that proteins upon binding with silica undergo mobility changes. Significance of the existence of silica binding protein in plasma and lung of rat in relation to silica toxicity is discussed.     

Direct enantiospecific HPLC bioanalysis of (R,S)-atenolol on a chiral stationary phase

The simultaneous determination of the enantiomers of the β₁-selective adrenergic antagonist atenolol in human plasma and urine is described. After an alkaline preextraction atenolol is extracted from biological material at pH 12.3 using dichloromethane/propan-2-ol. The separation of the underivatized enantiomers is achieved by high-performance liquid chromatography on a chiral stationary phase (Chiralcel OD, cellulose tris-3, 5-dimethylphenylcarbamate, coated on silica gel) with fluorimetric detection. (?)-(S)-Pindolol is used as an internal standard. The detection limits of 5 ng/ml enantiomer in plasma and 50 ng/ml enantiomer in urine are sufficient for pharmacokinetic studies after therapeutic doses. © 1993 Wiley-Liss, Inc.     

Gas chromatography-mass spectrometry characterisation of the anti-Listeria components of Garcinia kola seeds

Adsorption chromatography was used to separate the bioactive constituents of the crude n-hexane extract of Garcinia kola seeds. The silica gel 60 column fractions were eluted using the solvent combination of benzene:ethanol:ammonium hydroxide (BEA) in the ratio combination of 36:4:0.4 v/v. The fractions were tested for anti-Listeria activities by determining their MIC₅₀, MIC₉₀ or MIC against 4 Listeria isolates. The fractions were labelled BEA1 to BEA5 and 3 out of the 5 fractions eluted were active against the test Listeria species with MIC’s ranging from MIC 0.157 mg/mL to MIC₅₀ 0.625 mg/mL. The most active fractions, BEA2 and BEA3, were subjected to gas chromatography coupled to mass spectrometry (GC-MS) to identify their composition. Fraction BEA2 constituted of 18 compounds mostly sterols and the BEA3 fraction contained 27 compounds with the most abundant compounds being fatty acids derivatives. The BEA2 fraction’s interactions with antibiotics proved to be 100% synergistic with ciprofloxacin and ampicillin whilst it exhibited 50% additivity and 50% synergism with penicillin G. However, all the interactions of the BEA2 fraction with each of the conventional antibiotics used were synergistic against the human listeriosis causative bacteria Listeria monocytogenes.     

Isolation and identification of ferrioxamine G and E inHafnia alvei

Summary Under conditions of iron-deprivationHafnia alvei (Enterobacteriaceae) produces ferrioxamine G as the principal siderophore. Maximum hydroxamate siderophore production occurred at medium iron limitation. The ferrioxamines were extracted, purified by gel filtration and chromatography on silica gel yielding a major and a minor siderophore fraction. The minor siderophore fraction contained three siderophores, among which ferrioxamine E could be identified by HPLC and FAB mass spectrometry. Reductive hydrolysis of the ferrioxamine G fraction yielded succinic acid and a mixture of diaminopentane and diaminobutane, as determined by gas-liquid chromatography and GLC/MS. HPLC and FAB mass spectrometry confirmed that the ferrioxamine G fraction consisted of two different species, G₁ and G₂, possessing molecular masses of 671 Da and 658 Da respectively.     

Consecutive analysis of sphingoglycolipids on the basis of sugar and ceramide moieties by high performance liquid chromatography

A quantitative consecutive method was developed for analysis of sphingoglycolipids in biological materials by high performance liquid chromatography (HPLC). Crude lipid extracts were separated into neutral and acidic fractions on a DEAE-Sephadex column. Glycolipid fractions were obtained by acetylation and Florisil column chromatography, and the acetylated glycolipids were N-p-nitrobenzoylated by treatment with p-nitrobenzoyl chloride in pyridine at 60 degrees C for 6 h. Excess reagent and by-products were removed by solvent partition and gel filtration. The glycolipid derivatives were analyzed by their absorption at 254 nm on Zorbax SIL, a silica gel column, with a gradient of 0.5--7% isopropanol in hexane-chloroform (2 : 1, v/v) at a flow rate of 0.5 ml/min. The detector response was linear with up to 60 nmol of injected glycolipids. The practical lower limit of detection was about 50 pmol. The derivatives were separated on the basis of their sugar chains. Effluents corresponding to each peak were collected and analyzed further on the basis of their lipid portion on mu-Bondapak C18, a reversed phase column. This combined procedure was applied to the analysis of erythrocyte glycolipids. Samples containing as little as 20 micrograms of glycolipids could be analyzed by this method.     

Endogenous inotropic substance from heart tissue has digitalis-like properties.

In the past few years, we developed an extraction procedure which we successfully used to isolate a crude fraction containing digitalis-like substance (DLS) from porcine left ventricular tissue. In this study, the crude fraction was found to cross-react with digoxin antibodies and showed immunoreactivity of 4.25 +/- 0.6 ng digoxin equivalent/ml. On further purification of the crude fraction using silica gel G column chromatography, a fraction C was obtained, which was highly positive inotropic on canine trabeculae and it dose-dependently inhibited ouabain sensitive 86Rb+ uptake in rat heart slices. A 50% inhibition of uptake was obtained by 25 microliters of fraction C. Fraction C also inhibited canine kidney Na+, K(+)-ATPase (Sigma, U.S.A.) dose-dependently and a 50% inhibition of this enzyme required 17 microliters of fraction C. Ashing of the fraction C at 500 degrees C resulted in loss of inotropic and enzyme inhibitory activities, indicating an organic nature of the unknown digitalis-like substance.     

Isolation and characterization of potent bioactive fraction with antioxidant and UV absorbing activity from Aloe barbadensis Miller gel

The methanolic extract of Aloe vera L. gel was subjected to antioxidant guided fractionation with silica gel column chromatography to screen the potent fraction. The antioxidant capacities of different fractions were evaluated using DPPH radical scavenging assay and correlated with total phenol content. Total phenolic contents of different fractions were determined according to the Folin-Ciocalteu spectrophotometric method. The positive correlation was observed between DPPH radical scavenging assay and total phenolic contents indicating that phenolics in Aloe vera L. gel were fundamental contributor of antioxidant activity. Third pooled fraction was identified as potential fraction with highest antioxidant potential. This fraction indicated the presence of well resolved fluorescent components. Characteristic UV–vis absorption and HPLC analysis indicates that aloin take part in antioxidant potential attributed to aloe gel.     

Induction of tryptophan oxygenase and tyrosine aminotransferase by metabolites of hydrocortisone

Metabolites of hydrocortisone were isolated from rat liver on a preparative scale, fractionated by column chromatography on Sephadex Lh-20 and silica gel and tested for biological activity. Apart from the well known neutral metabolites, steroid glucuronides and sulfates, we obtained metabolite fractions containing non-conjugated steroidal carboxy acids and acid metabolites of unknown structure. One of these fractions induced tyrosine aminotransferase (EC 2.6.1.5) in adrenalectomized female rats but not tryptophan oxygenase (EC 1.13.11.11), whereas another one mainly increased activity of tryptophan oxygenase. The doses necessary to significantly induce both enzymes were much lower in case of these metabolites than in the case of hydrocortisone itself. The active fractions eluting from silica gel column were analyzed by thin-layer chromatography in two different solvent systems. Absence of hydrocortisone in these fractions could be clearly demonstrated. Furthermore, the active fractions eluting from the silica gel column were characterized by treatment with an extract from Helix pomatia and/or diazomethane and subsequent analysis by thin-layer chromatography. We conclude, considering the biological activity of some synthesized derivatives of hydrocortisone, that the biologically active components are acid metabolites of hydrocortisone which are not identical to any of the known metabolites.     

Rapid isolation of vesicular and micellar carriers of biliary lipids by ultracentrifugation

A simple, rapid, and new method has been developed to isolate and to quantitate the vesicular carrier of biliary lipids by isopycnic ultracentrifugation. The method combines the use of Metrizamide, as an inert centrifugation media to change the density of bile for isopycnic separation of vesicles, and a vertical rotor, to decrease both the time of centrifugation and the pressure of the hydrostatic column in the ultracentrifuge tube. Vesicles harvested from bile-Metrizamide density gradients were identified by negative staining electron microscopy. The buoyant densitites of biliary vesicles varied between 1.010 and 1.030 g/ml. The diameter of vesicles in fractions with d less than 1.020 g/ml was 82 +/- 10 nm and in fraction with d approximately 1.030 g/ml was 57 +/- 8 nm. Gel filtration chromatography with Ultrogel AcA 34 was used to validate the quantitive isolation of vesicles by the ultracentrifugal method. In experiments with bile-Metrizamide continuous preformed density gradients, greater than 93% of vesicular cholesterol was found in fractions with d less than 1.030 g/ml after 285 min of centrifugation at 50,000 rpm in a VTi vertical rotor (Beckman Instruments, Inc.). When 16% Metrizamide was dissolved in bile and centrifuged for 120 min, greater than 96% of total vesicular cholesterol was found in the top 0.4 ml of the 5-ml centrifuge tube, as assessed by gel filtration chromatography. This fraction contained less than 8% of cholesterol carried in micelles, as assessed by gel filtration chromatography. The variation coefficient of this short ultracentrifugal method to isolate biliary vesicles was 4.6%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of guanine nucleotides and protein kinase C on prolactin-stimulated release of Ca from intracellular stores of pig oocytes

The effects of guanine nucleotides and protein kinase C on prolactin-stimulated Ca2+ release from intracellular stores of pig oocytes were studied using the fluorescent dye chlorotetracycline. The effect of prolactin was related to the protein kinase C activation. Inhibition of protein kinase C stimulated Ca2+ release from intracellular stores of the pig oocytes treated with 5 ng/ml prolactin in the presence of extracellular Ca2+ and inhibited Ca2+ release from intracellular stores of the pig oocytes treated with 50 ng/ml prolactin. In a Ca2+-free medium, prolactin did not stimulate Ca2+ release from intracellular stores of the oocytes treated with GDP in the presence of GDP. GTP inhibition of protein kinase C activated Ca2+ release from intracellular stores of the pig oocytes treated with 5 ng/ml prolactin and inhibited Ca2+ release from intracellular stores of the pig oocytes treated with 50 ng/ml prolactin. These data suggest the influence of guanine nucleotides and protein kinase C on calcium metabolism, stimulated by prolactin.     

Bioactivity-guided fractionation for analgesic properties and constituents of Vitex negundo L. seeds

This study was undertaken to ascertain the analgesic properties of Vitex negundo L. seeds and to isolate and characterize the active constituents. Among the 80% ethanol extract and some fractions with different polarity, the acetoacetate fraction showed the highest anti-nociceptive activity in acetic acid-induced writhing test in ICR mice. The analgesic bioguided isolation of the acetoacetate fraction yielded two major lignans: 6-hydroxy-4-(4-hydroxy-3-methoxy-phenyl)-3-hydroxymethyl-7-methoxy-3, 4-dihydro-2-naphthaldehyde (1) and vitedoamine A (2). Given orally, compound (1), which was more productive, produced significant inhibitions on chemical nociception induced by intraperitoneal acetic acid and subplantar formalin injections and exhibited notable anti-inflammatory activities in dimethyl benzene-induced ear edema test in a dose-dependent manner. Since co-administration of naloxone fails to antagonize the analgesic activity of compound (1) in the formalin test, we suggest that compound (1) possesses potent analgesic effects which are most likely to be mediated by its anti-inflammatory activity rather than through opioid receptor system and therefore could partially explain the anti-nociceptive effect of V. negundo L. seeds.