Isolation and on-line identification of antioxidant compounds from three Baccharis species by HPLC-UV-MS/MS with post-column derivatisation

The aqueous extracts of aerial parts of Baccharis trimera (Less.) DC., B. crispa Spreng. and B. usterii Heering (Asteraceae) displayed significant radical scavenging activity in a diphenylpicrylhydrazole (DPPH)/TLC assay. In order to rapidly identify the active principles, the crude extracts were analysed by HPLC-UV, and an HPLC-micro-fractionation of the extract of B. usterii was performed. Six quinic acids derivatives (1-6) were isolated from B. usterii by MPLC. The fractions were monitored by DPPH/TLC assay and a series of radical-scavenging quinic acid derivatives could be identified. The comparison of the HPLC profiles of the extracts of B. usterii, B. trimera and B. crispa was performed. In order to obtain complementary on-line structural information for all peaks of interest, HPLC-MS/MS together with HPLC-UV involving post-column addition of UV shift reagents was undertaken on the crude extract. The interpretation of these data permitted the on-line identification of known compounds, some of which are reported for the first time in this plant.     

Enzyme inhibition activities of Andrachne cardifolia Muell

The crude methanolic extract and various fractions of Andrachne cardifolia Muell, including chloroform, ethyl acetate and n-butanol fractions were subjected to in vitro enzyme inhibition activity against acetylcholinesterase, butyrylcholinesterase, lipoxygenase and urease enzymes. A significant enzyme inhibition activity (40-89%) was shown by the crude methanolic extract and its fractions against lipoxygenase, while low to significant activity (40-71%) against butyrylcholinesterase. The crude methanolic extract and its various fractions demonstrated poor to significant activity (25-73%) against acetylcholinesterase and no activity against urease.     

Iridoid glycosides from the stems of Pithecoctenium crucigerum (Bignoniaceae)

Sixteen crude extracts from six Panamanian plants of the family Bignoniaceae were submitted to rapid TLC tests against DPPH and acetylcholinesterase. Pithecoctenium crucigerum (L.) A.H. Gentry, which showed interesting activity against DPPH, has been studied. The chemical investigation of the methanol extract from the stems afforded the iridoid glycoside theviridoside and three derivatives (6'-O-cyclopropanoyltheviridoside, 10-O-hydroxybenzoyltheviridoside and 10-O-vanilloyltheviridoside), along with five known phenylethanoid glycosides (verbascoside, isoverbascoside, forsythoside B, jionoside D and leucosceptoside B). These last compounds were all active against DPPH. The structures were determined by means of spectrometric and chemical methods, including 1D and 2D NMR experiments and MS analysis.     

Enzyme inhibition activities of Teucrium royleanum

The crude methanolic extract and chloroform, ethyl acetate and n-butanol fractions of Teucrium royleanum were examined as inhibitors of actylcholinesterase, butyrylcholinesterase, lipoxygenase and urease. A significant enzyme inhibition activity (52-83%) was shown by the crude methanolic extract and its fractions against acetylcholinesterase, while low to outstanding enzyme inhibitory activity was shown (19-93%) against butyrylcholinesterase. The crude methanolic extract and its various fractions demonstrated low activity against lipoxygenase and inactive against urease.     

Enzyme inhibition activities of teucrium royleanum

The crude methanolic extract and chloroform, ethyl acetate and n-butanol fractions of Teucrium royleanum were examined as inhibitors of actylcholinesterase, butyrylcholinesterase, lipoxygenase and urease. A significant enzyme inhibition activity (52–83%) was shown by the crude methanolic extract and its fractions against acetylcholinesterase, while low to outstanding enzyme inhibitory activity was shown (19–93%) against butyrylcholinesterase. The crude methanolic extract and its various fractions demonstrated low activity against lipoxygenase and inactive against urease.     

Lipoxygenase inhibition by novel fatty acid ester from Annona squamosa seeds

Studies on the seeds of Annona squamosa yielded a novel lipoxygenase inhibitor fatty acid ester, (+) - annonlipoxy (1). Compound 1 was screened for its enzyme inhibitory activity against lipoxygenase (E.C.1.14.18.1), exhibiting activity with IC(50) 69.05 +/- 5.06 microm. Baicalein (IC(50) 22.6 +/- 0.5 microm) was used as a positive control. Crude extracts of Annona squamosa fruit pulp and seeds were screened for its enzyme inhibitory activity against lipoxygenase and acetylcholinesterase. The crude ethanolic extract of fruit pulp and seeds of Annona squamosa also exhibited lipoxygenase activity with 22.2 and 26.7% inhibition, while the pet.ether extract of seeds of A. squamosa exhibited 52.7% inhibition at a concentration of 40 microg/200 ml. The crude ethanolic extract of seeds of Annona squamosa was also bioassayed for acetylcholinesterase inhibition and it was found inactive.     

Inhibition activities of colchicum luteum baker on lipoxygenase and other enzymes

In vitro enzymes inhibition activities of the crude methanolic extract and various fractions of Colchicum luteum Baker (Liliaceae) including chloroform, ethyl acetate, n-butanol and aqueous were carried out against actylcholinesterase, butyrylcholinesterase, lipoxygenase and urease enzymes. A significant enzyme inhibition activity (89%) is shown by the crude methanolic extract and its fractions against lipoxygenase, while low to significant activity (32–75%) was evident against butyrylcholinesterase. The crude methanolic extract and its various fractions demonstrated low activity (29–61%) against acetylcholinesterase and no activity against urease.     

Inhibition activities of Colchicum luteum baker on lipoxygenase and other enzymes

In vitro enzymes inhibition activities of the crude methanolic extract and various fractions of Colchicum luteum Baker (Liliaceae) including chloroform, ethyl acetate, n-butanol and aqueous were carried out against actylcholinesterase, butyrylcholinesterase, lipoxygenase and urease enzymes. A significant enzyme inhibition activity (89%) is shown by the crude methanolic extract and its fractions against lipoxygenase, while low to significant activity (32-75%) was evident against butyrylcholinesterase. The crude methanolic extract and its various fractions demonstrated low activity (29-61%) against acetylcholinesterase and no activity against urease.     

Allelopathic effects of water hyacinth [Eichhornia crassipes

Eichhornia crassipes (Mart) Solms is an invasive weed known to out-compete native plants and negatively affect microbes including phytoplankton. The spread and population density of E. crassipes will be favored by global warming. The aim here was to identify compounds that underlie the effects on microbes. The entire plant of E. crassipes was collected from El Zomor canal, River Nile (Egypt), washed clean, then air dried. Plant tissue was extracted three times with methanol and fractionated by thin layer chromatography (TLC). The crude methanolic extract and five fractions from TLC (A-E) were tested for antimicrobial (bacteria and fungal) and anti-algal activities (green microalgae and cyanobacteria) using paper disc diffusion bioassay. The crude extract as well as all five TLC fractions exhibited antibacterial activities against both the gram positive bacteria; Bacillus subtilis and Streptococcus faecalis; and the gram negative bacteria; Escherichia coli and Staphylococcus aureus. Growth of Aspergillus flavus and Aspergillus niger were not inhibited by either E. crassipes crude extract nor its five fractions. In contrast, Candida albicans (yeast) was inhibited by all. Some antialgal activity of the crude extract and its fractions was manifest against the green microalgae; Chlorella vulgaris and Dictyochloropsis splendida as well as the cyanobacteria; Spirulina platensis and Nostoc piscinale. High antialgal activity was only recorded against Chlorella vulgaris. Identifications of the active antimicrobial and antialgal compounds of the crude extract as well as the five TLC fractions were carried out using gas chromatography combined with mass spectroscopy. The analyses showed the presence of an alkaloid (fraction A) and four phthalate derivatives (Fractions B-E) that exhibited the antimicrobial and antialgal activities.     

Presence of cholinomimetic and acetylcholinesterase inhibitory constituents in betel nut

In this investigation, we report the presence of cholinomimetic and acetylcholinesterase (AChE) inhibitory constituents in betel nut, the most commonly used drug in the world after tobacco, ethanol and caffeine. The crude extract of betel nuts or Areca catechu (Ac.Cr) caused a dose-dependent (0.3-300 microg/mL) spasmogenic effect in the isolated rabbit jejunum. The spasmogenic effect was blocked by atropine, similar to that of acetylcholine (ACh), suggestive of muscarinic receptor mediated effect. Both the extract (0.3-10 microg/mL) and physostigmine (0.1-3.0 microM) potentiated the effect of a fixed dose of ACh (10 microM) in a dose-dependent fashion, suggesting acetylcholinesterase (AChE) inhibitory effect. This effect was confirmed in the in vitro assay where both the crude extract (1-100 microg/mL) and physostigmine inhibited the enzyme. In the in vivo model of gastrointestinal transit, Ac.Cr (10-30 mg/kg) enhanced the travel of charcoal meal and also exhibited a laxative effect in mice. The plant extract was subjected to activity-directed fractionation and all resultant fractions showed atropine-sensitive spasmogenicity in rabbit jejunum and also AChE inhibitory effect at doses similar to that for the parent crude extract, the ethyl acetate fraction being slightly less potent. Some of the known constituents of betel nut, including arecoline, were tested for the possible inhibitory effect on AChE, none were found active. The study provides first evidence for the presence of AChE inhibitory constituents in betel nut, though additional direct muscarinic stimulatory effect cannot be ruled out and this study provides sound scientific basis for some of the folkloric uses associated with betel nut chewing.     

Lipoxygenase inhibition by novel fatty acid ester from Annona squamosa seeds

Studies on the seeds of Annona squamosa yielded a novel lipoxygenase inhibitor fatty acid ester, (+) - annonlipoxy (1). Compound 1 was screened for its enzyme inhibitory activity against lipoxygenase (E.C.1.14.18.1), exhibiting activity with IC₅₀ 69.05 ± 5.06 μm. Baicalein (IC₅₀ 22.6 ± 0.5 μm) was used as a positive control. Crude extracts of Annona squamosa fruit pulp and seeds were screened for its enzyme inhibitory activity against lipoxygenase and acetylcholinesterase. The crude ethanolic extract of fruit pulp and seeds of Annona squamosa also exhibited lipoxygenase activity with 22.2 and 26.7% inhibition, while the pet.ether extract of seeds of A. squamosa exhibited 52.7% inhibition at a concentration of 40 μg/200 ml. The crude ethanolic extract of seeds of Annona squamosa was also bioassayed for acetylcholinesterase inhibition and it was found inactive.     

Enzyme inhibition activities of the extracts from rhizomes of Gloriosa superba Linn (Colchicaceae)

An alcoholic extract obtained from the rhizomes of Gloriosa superba Linn (Colchicaceae) was screened for enzyme inhibition activities. The crude extract and its subsequent fractions including chloroform, ethyl acetate, n-butanol and aqueous were screened against lipoxygenase, actylcholinesterase, butyrylcholinesterase and urease. An outstanding inhibition on lipoxygenase was observed. The highest enzyme inhibition potency was expressed by the chloroform fraction (90%) among the tested fractions on lipoxygenase. Overall 67-90% inhibition was found for lipoxygenase, 46-69% for acetylcholinesterase and 10-33% for butyrylcholinesterase, while urease was not inhibited.     

Antifungal efficacy of Thevetia peruviana leaf extract against Alternaria solani and characterization of novel inhibitory compounds by Gas Chromatography-Mass Spectrometry analysis

Alternaria solani, a plant pathogenic fungus causes significant economical losses of potato crop. The disease is controlled primarily through some traditional methods and most commonly via the application of chemical fungicides. Fungicides treatment is not protected as chemicals pollute environment, effect health vulnerability in humans and when these harmful chemicals enter into the food chain become hazardous to all living entities. Recent efforts have focused on developing environmentally safe, long-lasting, and effective biocontrol methods for the management of plant diseases. Present research focus on screening of crude and partially purified leaf extract of Thevetia peruviana for the presence of antifungal efficacy against Alternarai solani. It was observed that 100% alcoholic crude and alcoholic fraction of partially purified extract showed maximum inhibitory activity which is due to the presence of different secondary metabolites, revealed by phytochemical screening. Active column fraction (possess best antifungal activity against Alternaria solani) was subjected to Gas Chromatography-Mass Spectrometry (GS-MS) analysis. On the basis of peaks matching of GC-MS chromatogram with available data base showed the presence of benzoic acid and oxo-benzoate in active fraction of Thevetia peruviana leaf extract which is already known chemical among the phytochemicals described for antimicrobial activity. Further research on development of herbal formulation from the same would be very helpful environment friendly approach to manage concern crop disease.     

Characterization of an endophytic whorl-forming Streptomyces from Catharanthus roseus stems producing polyene macrolide antibiotic

An endophytic whorl-forming Streptomyces sp. designated as TS3RO having antifungal activity against a large number of fungal pathogens, including Sclerotinia sclerotiorum, Rhizoctonia solani, Colletotrichum gloeosporioides, Cryphonectria parasitica, Fusarium oxysporum, Pyrenophora tritici-repentis, Epidermophyton floccosum, and Trichophyton rubrum, was isolated from surface-sterilized Catharanthus roseus stems. Preliminary identification showed that Streptomyces cinnamoneus subsp. sparsus was its closest related species. However, strain TS3RO could readily be distinguished from this species using a combination of phenotypic properties, 16S rDNA sequence similarity, and phylogenetic analyses. Thus, the whorl-forming Streptomyces sp. strain TS3RO is likely a new subspecies within the Streptomyces cinnamoneus group. Direct bioautography on a thin-layer chromatography plate with Cladosporium cucumerinum was conducted throughout the purification steps for bioassay-guided isolation of the active antifungal compounds from the crude extract. Structural elucidation of the isolated bioactive compound was obtained via LC-MS spectrometry, UV-visible spectra, and nuclear magnetic resonance data. It revealed that fungichromin, a known methylpentaene macrolide antibiotic, was the main antifungal component of TS3RO strain, as shown by thin-layer chromatography bioautography. This is the first report of an endophytic whorl-forming Streptomyces isolated from the medically important plant Catharanthus roseus.     

Monoclonal Antibodies to Rat Brain Acetylcholinesterase: Comparative Affinity for Soluble and Membrane-Associated Enzyme and for Enzyme from Different Vertebrate Species

Seven unique monoclonal antibodies were generated to rat brain acetylcholinesterase. Upon density gradient ultracentrifugation, immunoglobulin complexes with the monomeric enzyme appeared as single peaks of acetylcholinesterase activity with a sedimentation coefficient approximately 3S greater than that of the free enzyme. This behavior is consistent with the assumption of one binding site per enzyme molecule. Apparent dissociation constants of these antibodies for rat brain acetylcholinesterase calculated on the basis of this assumption ranged from about 10 nM to more than 1,000 nM. Some of the antibodies were less able to bind the membrane-associated enzyme that required detergent for solubilization than the naturally soluble acetylcholinesterase of detergent-free brain extracts. Species cross-reactivity was investigated with crude brain extracts from mammals (human, mouse, rabbit, guinea pig, cow, and cat) and from other vertebrates (chicken, frog, and electric eel). Three antibodies bound rat acetylcholinesterase exclusively; one had nearly the same affinity for all mammalian acetylcholinesterases investigated; the remaining three showed irregular binding patterns. None of the antibodies recognized frog and electric eel enzyme. Pooled antibody was found to be suitable for specific immunofluorescence staining of large neurons in the ventral horn of the rat spinal cord and smaller cells in the caudate nucleus. Other potential applications of these antibodies are discussed.     

HPLC coupled on-line to ESI-MS and a DPPH-based assay for the rapid identification of anti-oxidants in Butea superba

A reversed-phase HPLC coupled on-line to a radical scavenging detection system and MS/MS was developed in order to combine separation, activity determination and structural identification of anti-oxidants in complex mixtures in one run. The sample was separated by HPLC and the eluate split into two flows. The major portion was fed into an electrospray ionisation MS/MS system, while the minor part was mixed with a free radical, 2,2'-diphenyl-1-picrylhydrazyl (DPPH), and the reaction determined spectrophotometrically. The negative peaks, which indicated the presence of anti-oxidant activity, were monitored by measuring the decrease in absorbance at 517 nm. The developed method was successfully applied to the identification of anti-oxidant compounds in a fraction, obtained by solid-phase extraction, of an extract of a Thai medicinal plant, Butea superba Roxb. The anti-oxidant compounds were separated and identified as procyanidin B2, (-)-epicatechin and procyanidin B5.     

Screening for acetylcholinesterase inhibitory activity in cyanobacteria of the genus Nostoc

Fifty-four cyanobacterial strains of the genus Nostoc from different habitats were screened for acetylcholinesterase inhibitory activity. Water-methanolic extracts from freeze-dried biomasses were tested for inhibitory activity using Ellman's spectrophotometric method. Acetylcholinesterase inhibitory activity higher than 90% was found in the crude extracts of Nostoc sp. str. Lukesova 27/97 and Nostoc ellipsosporum Rabenh. str. Lukesova 51/91. Extracts from Nostoc ellipsosporum str. Lukesova 52/91 and Nostoc linckia f. muscorum (Ag.) Elenk. str. Gromov, 1988, CALU-980 inhibited AChE activity by 84.9% and 65.3% respectively. Moderate AChE inhibitory activity (29.1-37.5%) was found in extracts of Nostoc linckia Roth. str. Gromov, 1962/10, CALU-129, Nostoc muscorum Ag. str. Lukesova 127/97, Nostoc sp. str. Lhotsky, CALU-327 and Nostoc sp. str. Gromov, CALU-998. Extracts from another seven strains showed weak anti-AChE activities. The active component responsible for acetylcholinesterase inhibition was identified in a crude extract of Nostoc sp. str. Lukesova 27/97 using HPLC and found to occur in one single peak.     

Enzyme inhibition activities of the extracts from rhizomes of Gloriosa superba Linn (Colchicaceae)

An alcoholic extract obtained from the rhizomes of Gloriosa superba Linn (Colchicaceae) was screened for enzyme inhibition activities. The crude extract and its subsequent fractions including chloroform, ethyl acetate, n-butanol and aqueous were screened against lipoxygenase, actylcholinesterase, butyrylcholinesterase and urease. An outstanding inhibition on lipoxygenase was observed. The highest enzyme inhibition potency was expressed by the chloroform fraction (90%) among the tested fractions on lipoxygenase. Overall 67– 90% inhibition was found for lipoxygenase, 46-69% for acetylcholinesterase and 10–33% for butyrylcholinesterase, while urease was not inhibited.     

Chemical structure and antiviral activity of carrageenans from Meristiella gelidium against herpes simplex and dengue virus

Chemical and spectroscopic methods showed that the major KCl-precipitated galactans from Meristiella gelidium (Solieriaceae) are iota/kappa/nu-hybrid carrageenans with the former one in higher proportion. These carrageenans showed, by HPSEC-MALLS analysis, unimodal symmetrical peaks with MW of 425.6–956.7 kDa. The effectiveness of the crude extracts from M. gelidium against HSV-2 was higher than the corresponding extract from G. griffithsiae, previously determined. However, when considering the homogeneous carrageenans, the fractions obtained from both seaweeds showed the same level of activity. The extracts and carrageenan derived from M. gelidium were more effective inhibitors of DENV-2 if compared with G. griffithsiae samples and reference polysaccharides. The most active fraction obtained from M. gelidium showed a selectivity index against HSV-2 of 25,000, a value high enough to consider this carrageenan as a promising agent to be evaluated for the treatment of genital HSV-2 infections.     

Separation in a single step by affinity chromatography of cholinesterases differing in subunit number.

We describe an affinity chromatography method in which dimethylaminoethylbenzoic acid-Sepharose 4B is used, making it possible to separate in one step the molecular forms of globular acetylcholinesterase (AChE, EC 3.1.1.7) or butyrylcholinesterase (ChE, EC 3.1.1.8). A crude extract containing these enzymes was deposited onto the chromatography gel, washed, and eluted by a linear gradient of tetramethylammonium chloride (0-0.3 M). With rat brain AChE, two well-separated peaks were eluted in the presence of 1% Triton X-100; the first peak corresponded to 4 S forms and the second to 11 S forms. This separation was very efficient for salt-soluble activity and less efficient for the detergent-soluble AChE. In this case, the 4 S peak represented only 6.5% of total detergent-soluble activity and was cross-contaminated by the 11 S form. Rat serum ChE was efficiently separated into two peaks of 7 S and 11 S. This method could potentially be adapted to separate other multimeric proteins with varying numbers of affinity sites.