Bioassay-guided Isolation Procedure Research Articles

Bioassay-Guided Isolation of an Abetiane-Type Diterpenoid from Prunella vulgaris That Protects against Concanavalin A-Induced Autoimmune Hepatitis.

Prunella vulgaris is a widely used edible Chinese medicinal plant. In the present study, two new abietane-type diterpenoids, abietoquinones A (1) and B (2), were isolated from this plant by an immunosuppressive bioassay-guided isolation procedure. Their structures were elucidated unambiguously by NMR spectroscopic analysis, single-crystal X-ray crystallography, and electronic circular dichroism calculations. Compounds 1 and 2 bear a cyclohex-2-ene-1,4-dione moiety, which is uncommon among abietane diterpenes. Also, abietoquinone A (1) suppressed murine splenocyte proliferation and decreased the production of proinflammatory cytokines induced by concanavalin A (Con A) in vitro. In Con A-challenged mice, preinjection with 1 significantly ameliorated liver injury. Additionally, abietoquinone A (1) exhibited inhibitory activities against the proliferation of murine splenocytes and human T cells induced by anti-CD3/anti-CD28 monoclonal antibodies (mAbs).

The In Vitro Anti-Cancer Activities of 17βH-Neriifolin Isolated from Cerbera odollam and its Binding Activity on Na+, K+-ATPase.

17βH-neriifolin, a cardiac glycoside compound had been successfully isolated from Cerbera odollam leaves based on the bioassay guided-isolation procedure. The aim of these studies were to determine the in vitro anti-cancer and binding effects of 17βH-neriifolin on Na+, K+-ATPase. The in vitro anti-cancer effects were evaluated using Sulphorhodamine B and Hoescht 33342 assays. The Na+, K+-ATPase assay was carried out using Malachite Green assay. In silico molecular docking studies and in vitro malachite green assay were used to predict the binding activities of 17βH-neriifolin on Na+, K+-ATPase and ouabain was also included as for comparison studies. The compound was tested against breast (MCF-7, T47D), colorectal (HT-29), ovarian (A2780, SKOV-3) and skin (A375) cancer cell lines that gave IC50 values ranged from 0.022 ± 0.0015 to 0.030 ± 0.0018 μM. The mechanism of cell death of 17βH-neriifolin was further evaluated using Hoescht 33342 assay and it was found that the compound killed the cancer cells via apoptosis. 17βHneriifolin and ouabain both bound at α-subunit in Na+, K+-ATPase and their binding energy were - 8.16 ± 0.74 kcal/mol and -8.18 ± 0.48 kcal/mol respectively. The results had confirmed the anti-proliferative effects exerted by 17βH-neriifolin in the breast, colorectal, ovarian and skin cancer cell lines. 17βH-neriifolin had shown to cause apoptotic cell death in the respective cancer cell lines.17βH-neriifolin and ouabain both bound at α-subunit in Na+, K+-ATPase and their binding energy were -8.16 ± 0.74 kcal/mol and -8.18 ± 0.48 kcal/mol respectively. This is the first report to reveal that 17βH-neriifolin managed to bind to the pocket of α-subunit of Na+.K+-ATPase.

Bacillus methylotrophicus ASWU-C2, a strain inhabiting hot desert soil, a new source for antibacterial bacillopyrone, pyrophen, and cyclopeptides.

A strain of Bacillus methylotrophicus was isolated from a soil sample collected in Aswan eastern desert, which is known for its extremely arid climate. After fermentation of the strain in liquid culture and subsequent extraction, a bioassay-guided isolation procedure yielded five compounds: 2-benzyl-4H-pyran-4-one, named bacillopyrone (1), pyrophen (2), macrolactin A (3) and the cyclopeptides malformin A1 (4), and bacillopeptin A (5). The structures were determined by interpretation of nuclear magnetic resonance (NMR) spectroscopy and high resolution mass spectrometry (HR-MS) data. This is the first report on the isolation of compounds 1 and 2 from Bacillus species; compound 1 was reported previously as synthetic product. Bacillopyrone (1) exhibited moderate activity against the Gram-negative Chromobacterium violaceum with minimum inhibitory concentration 266.6 μg/mL, while macrolactin A (3) and malformin A1 (4) inhibited Staphylococcus aureus (minimum inhibitory concentrations 13.3 and 133.3 μg/mL, respectively).

Isolation and characterization of a new cytotoxic dihydrophenanthrene from Dioscorea membranacea rhizomes and its activity against five human cancer cell lines

Ethnopharmacological relevanceThe rhizomes of Dioscorea membranacea Pierre (DM) have been used as ingredients in anticancer herbal formulations in Thai traditional medicine (TTM). Thus, the aim of this study was to investigate the active constituents of DM for cytotoxic activity in order to support its TTM use. Materials and methodsA bioassay-guided isolation procedure was used to separate the cytotoxic constituents from ethanolic extract of Dioscorea membranacea rhizomes by testing against five human cancer cell lines, i.e. large cell lung carcinoma, COR-L23; liver cancer cells, HepG2; prostate cancer cells, PC3; breast cancer cells MCF-7; cervical cancer cells, Hela; and one normal human lung cell line (MRC 5) using the SRB assay. ResultsTwo known dihydrophenanthrene compounds [2,4 dimethoxy-5,6-dihydroxy-9,10-dihydrophenanthrene (1) and 5-hydroxy-2,4,6-trimethoxy-9,10-dihydrophenanthrene (2)], and a new dihydrophenanthrene compound, 5,6,2 -trihydroxy 3,4-methoxy, 9,10-dihydrophenanthrene (3) were isolated and fully characterized. 1 showed the highest cytotoxic activity against COR-L23, MCF-7 and PC3 cell lines (IC50=14.89, 17.49 and 19.04µM, respectively), and 2 showed selective cytotoxic activity against PC3 (IC50=23.54µM). The new compound 3 showed selective cytotoxic activity against only MCF-7 cells (IC50=31.41µM). Interestingly the crude extract of DM was much less toxic to the normal cell line (MRC-5) (IC50>50µg/ml) compared to the five cancer cell lines, (IC50 value ranged between 6 and 29µg/ml). ConclusionThe phytochemicals isolated from DM may serve as lead compounds for the design of new anti-cancer agents with better selective cytotoxic indices.

A pair of chiral flavonolignans as novel anti‐cyanobacterial allelochemicals derived from barley straw (Hordeum vulgare): characterization and comparison of their anti‐cyanobacterial activities

The inhibitory effect of barley straw (Hordeum vulgare) on cyanobacteria has been observed in many field and laboratory studies for over 30 years, although the compounds responsible for this anti-cyanobacterial effect have remained unknown. In this study, a pair of chiral flavonolignans were isolated from barley straw extract using a bioassay-guided isolation procedure against Microcystis sp. The structures of the allelopathic compounds were elucidated by NMR (nuclear magnetic resonance) and HPLC-MS (high performance liquid chromatography-mass spectrometry), and turned out to be salcolin A and B. The enantiomers differ in their anti-cyanobacterial abilities. Both enantiomers exhibited inhibitory effects on Microcystis sp., and the EC50 (concentration for 50% of maximal effect) of salcolin A and B were 6.02 × 10(-5) and 9.60 × 10(-5 ) mol l(-1) , respectively. Furthermore, the modes of actions of the enantiomers were investigated and compared at a single cell level by flow cytometry. Salcolin A was found to induce an increase on cyanobacterial intracellular ROS (reactive oxygen species) levels and to inhibit esterase activity, whereas salcolin B caused leakages of cyanobacterial cytoplasms. Thus, salcolin A was more 'algistatic', and salcolin B was more 'algicidal'. This study suggests that salcolin is the key allelochemical in barley straw's inhibitory effect on cyanobacteria and could be used as an agent in the future control of cyanobacterial harmful algae blooms.

Traditional use, chemical composition and antimicrobial activity of Pectis brevipedunculata essential oil: A correlated lemongrass species in Brazil

For centuries, medicinal plants have been used as source of active principles for the treatment of many conditions. Ethnobotanical studies and bioassay guided isolation procedures have been successfully used in order to investigate and confirm their medicinal prescriptions. Traditionally used in Brazil, Cymbopogon citratus (lemongrass) is usually consumed as tea drink due to its calmative, anxyolitic as well as antihypertensive properties. Due to the similar lemongrass scent many species of Pectis genus have been used as infusion drinks for the same purposes as C. citratus. In Brazil, Pectis brevipedunculata, a sandy ornamental aromatic grass, is one of the “lemongrass odor” correlated species traditionally consumed. Chemical analysis of its essential oil was performed using GC-FID and GC-MS. Such essential oil was characterized by a high percentages of citral (81.8%: neral 35.6% and geranial 46.2%), followed by limonene (2.9%) and α-pinene (2.6%). Chemical and ethnobotanical investigations were performed involving one of the most commonly used Pectis species, known as lemongrass in order to confirm the medicinal indications compared to their chemical profile. The essential oil of P. brevipedunculata was tested against several clinical parasites. Our results were in agreement to the literature survey, suggesting the citral as the principal active constituent of the tested samples. Despite of the wide biological activities spectrum related to the major constituent presented in the essential oil of the most Pectis species, it is necessary to continue the phytochemical and pharmacological studies about the infusions constituents and validate the folk medicine.

Allium ursinum L.: Bioassay-Guided Isolation and Identification of a Galactolipid and a Phytosterol Exerting Antiaggregatory Effects

Aims: We wanted to investigate the possible antithrombotic effects and elucidate the chemical identity of the active principles involved in inhibitory effects against adenosine diphosphate (ADP)-induced aggregation of human platelets by wild garlic, Allium ursinum L. Methods: For this purpose, a bioassay-guided isolation procedure was used followed by spectrometric identification of pure active compounds. For the bioassay, blood was taken from healthy human volunteers and platelet-rich plasma was prepared for turbidimetric platelet aggregation tests. Platelet-rich plasma, stimulated with 20 µmol/l of ADP, was treated with extracts of different polarities, fractions and isolated single compounds from A. ursinum. The extracts were investigated by thin-layer chromatography (TLC), HPLC, mass spectroscopy, electrospray ionization mass spectrometry (ESI-MS) and 1/2-dimensional ¹H/¹³C-nuclear magnetic resonance (NMR) spectroscopic techniques. Results: Fresh A. ursinum leaves were extracted with ethanol, which was the potent form that effectively inhibited ADP-induced aggregation of human platelets. This ethanolic extract was subjected to liquid-liquid partition. Whilst the aqueous phase, containing the moiety of cysteine sulphoxide and thiosulphinate derivatives, showed only weak activity on platelet aggregation, the ethyl-acetate and particularly the chloroform partitions showed the highest aggregation-inhibiting potency. Thus, in our bioassay, the effects of alliins/allicins could be neglected. The chloroform phase, possessing the strongest activity, was separated into 28 fractions by gradient-elution open column chromatography on silica gel. The most active fractions 11–17 were separated again, yielding 10 subfractions. This afforded 1,2-di-O-α-linolenoyl-3-O-β-D-galactopyranosyl-sn-glycerol and β-sitosterol-3-O-β-D-glucopyranoside, the structures of which were determined by ESI-MS and 1/2-dimensional ¹H/¹³C NMR spectroscopic techniques. Furthermore, the minute amounts of volatile oil of A. ursinum leaves obtained by steam distillation according to Ph. Eur. could be evaluated as a third aggregation-inhibiting principle. Conclusion: In our study, for the first time, 2 active, non-sulphur-containing constituents of wild garlic, namely a galactolipid and a phytosterol, could be identified exhibiting inhibitory action on ADP-induced aggregation in human blood platelets. As a major constituent, the galactolipid, 1,2-di-O-α-linolenoyl-3-O-β-D-galactopyranosyl-sn-glycerol, not yet found in Allium sp., appears as a new, highly useful marker substance for A. ursinum drugs, or their pharmaceutical or food preparations, as shown by our orientating TLC analyses.

Inhibitory Effects of a Branched-Chain Fatty Acid on Larval Settlement of the Polychaete Hydroides elegans

Eleven strains of Streptomyces isolated from deep-sea sediments were screened for anti-larval settlement activity and all were active. Among those strains, Streptomyces sp. UST040711-290 was chosen for the isolation of bioactive antifouling compounds through bioassay-guided isolation procedure. A branched-chain fatty acid, 12-methyltetradecanoid acid (12-MTA) was purified, and it strongly inhibited the larval settlement of the polychaete Hydroides elegans. Streptomyces sp. UST040711-290 produced the highest yield of 12-MTA when the bacterium was cultured at 30 degrees C and pH 7.0 in a modified MGY medium. To investigate the potential antifouling mechanism of 12-MTA in the larval settlement of Hydroides elegans, the expression level of four marker genes, namely, Ran GTPase activating protein (GAP), ATP synthase (AS), NADH dehydrogenase (ND), and cell division cycle protein (CDC), was compared among the untreated larvae (the control), isobutylmethylxanthine (an effective settlement inducer), and 12-MTA-treated larvae. The 12-MTA treatment down-regulated the expression of GAP and up-regulated the expression of AS in the H. elegans larvae, but did not affect the expression of ND and CDC. This study provides the first evidence that a branched-chain fatty acid produced by a marine bacterium isolated from deep-sea sediment effectively inhibited the larval settlement of the biofouling polychaete H. elegans and its effects on the expression of genes important for larval settlement.

Insecticidal compounds of the biofilm‐forming cyanobacterium Fischerella sp. (ATCC 43239)

Cyanobacterial biofilms are grazed by many different benthic invertebrates. In particular, larvae of insects are often present in great numbers and exert strong grazing pressure on cyanobacteria. Along with morphological adaptations, allelochemicals may have been developed as defense mechanisms against insect larvae. To investigate the chemical defense of biofilm-forming cyanobacteria, larvae of Chironomus sp., a widely distributed genus in this habitat, were used. Ten artificial biofilms of axenic and nonaxenic cyanobacteria were screened for insecticidal activity against Chironomus sp. Fischerella 43239 was the cyanobacterium that exhibited the highest acute toxicity. A bioassay-guided isolation procedure was used to study the compounds responsible for toxicity in more detail. A toxic fraction was obtained when the 60% methanolic extract of Fischerella 43239 was separated on a C18 HPLC column. Electrospray mass spectrometry indicated the presence of several compounds in this fraction. The successful separation into individual compounds was achieved by HPLC on a cyanopropyl column. The heavily clustered quasimolecular ions observed on an electrospray mass spectrometer and the absorption spectra of the separated compounds were indicative of indole derivatives. The existence in the benthic cyanobacterium Fischerella 43239 of strong insecticidal metabolites that serve as chemical protection agents against insect larvae is supported by the data.

Oscillapeptin J, a new grazer toxin of the freshwater cyanobacterium Planktothrix rubescens.

Oscillapeptin J (1), a new and highly potent crustacean grazer toxin, was isolated from the axenic cyanobacterium Planktothrix rubescens, which frequently forms blooms in freshwater lakes. Chemical and spectroscopic analyses, including high resolving MS and two-dimensional NMR, were used to elucidate the compound's structure as a depsiheptapeptide of the oscillapeptin type. Strict application of a bioassay-guided isolation procedure proved this compound to be one of the major causative agents (besides [d-Asp(3),(E)-Dhb(7)]microcystin-RR) of the acute grazer toxicity of P. rubescens from Lake Zürich. The LC(50) value of oscillapeptin J as determined for the freshwater crustacean Thamnocephalus platyurus was 15.6 microM.

Integramides A and B, two novel non-ribosomal linear peptides containing nine C(alpha)-methyl amino acids produced by fungal fermentations that are inhibitors of HIV-1 integrase.

[structure: see text]. Integramides A and B are two novel 16-mer linear peptides rich in C(alpha)-methyl amino acids that were isolated from fungal extracts of Dendrodochium sp. by employing a bioassay-guided isolation procedure using recombinant HIV-1 integrase. The structure and stereochemistry were elucidated by a combination of 2D NMR and ESI- and FAB-MS including MS/MS studies and by Marfey's method. Integramides A and B inhibited the coupled reaction of HIV-1 integrase with IC50 values of 17 and 10 microM, respectively.

Antineoplastic Agents. 362. Isolation and X-ray Crystal Structure of Dibromophakellstatin from the Indian Ocean Sponge Phakellia mauritiana

Bioassay-guided isolation procedures using human tumor cell lines led to isolation of dibromophakellstatin (4) from the Republic of Seychelles sponge Phakellia mauritiana. The isolation, X-ray crystal structure elucidation, absolute stereochemistry, and antineoplastic activity have been summarized. P. mauritiana was also found to contain dibromophakellin (1), debromohymenialosine (2), thymidine, deoxyuridine, and thymine.

Antifungal polyoxygenated fatty acids fromAeollanthus parvifolius

Two polyoxygenated fatty acids with antifungal activity have been isolated from aerial parts ofAeollanthus parvifolius (Labiatae) using bioassay-guided isolation procedures. Spectroscopic analysis was used to identify the compounds as (Z)-4ξ,9ξ-diacetoxy-6ξ,7ξ-epoxy-5ξ-hydroxy-8-oxododeca-2-enoic acid and (Z)-4ξ,6ξ,7ξ,9ξ-tetraacetoxy-5ξ-hydroxy-8-oxododeca-2-enoic acid. Both of these compounds inhibited spore germination ofCladosporium cucumerinum. Minimum inhibitory doses were 1 µg and 5 µg, respectively, but only the former inhibited germination ofAspergillus niger at a dose of 5 µg. Neither compound exhibited antibacterial activity. (Z)-4ξ,9ξ-Diacetoxy-6ξ,7ξ-epoxy-5ξ-hydroxy-8-oxododeca-2-enoic acid reduced the hyphal growth ofPythium ultimum. The mode of action was not resolved but did not involve either the disruption of the cell wall membrane or the inhibition of extracellular enzymes. Application of (Z)-4ξ,6ξ,7ξ,9ξ-tetraacetoxy-5ξ-hydroxy-8-oxododeca-2-enoic acid to seedling plants did not give any protection against fungal attack, and indeed the compound appeared to be phytotoxic.

A New Antibacterial Sesquiterpene from Premna oligotricha

A novel sesquiterpene, 7 alpha-hydroxy-6, 11-cyclofarnes-3(15)-en-2-one [1], has been isolated from the aerial parts of Premna oligotricha (Verbenaceae) using an antimicrobial bioassay-guided isolation procedure. The sesquiterpene was identified on the basis of spectroscopic data and showed weak activity against Gram-positive bacteria Bacillus pumilus, Bacillus subtilis, Staphylococcus aureus, and Streptococcus faecalis.

Isolation and structure elucidation of novel hypotensive agents, niazinin A, niazinin B, niazimicin and niaziminin A + B from Moringa oleifera: the first naturally occurring thiocarbamates

Novel hypotensive principles, niazinin A 1, niazinin B 2, niazimicin 4 and niaziminin A and B 6+7, have been obtained from the ethanolic extract of the fresh leaves of Moringa oleifera, employing a bioassay-guided isolation procedure. Their structures have been elucidated through spectroscopic (including 2D NMR techniques) and chemical methods. These compounds are mustard oil glycosides which are very rare in Nature and are also the first examples of naturally occurring thiocarbamates.

A Novel Antibacterial Diterpene fromPremna schimperi

A novel diterpene, (5R, 8R, 9S, 10R)-12-oxo-ent-3,13(16)-clerodien-15-oic acid, has been obtained from the leaves of Premna schimperi (Verbenaceae) using an anti-microbial bioassay-guided isolation procedure. The diterpene was identified on the basis of spectroscopic data and is active against the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis in the concentration range 20-25 micrograms ml-1.