Bioassay-guided isolation and identification of antimicrobial compounds from thyme essential oil by means of overpressured layer chromatography, bioautography and GC-MS

A simple method is described for efficient isolation of compounds having an antibacterial effect. Two thyme (Thymus vulgaris) essential oils, obtained from the market, were chosen as prospective materials likely to feature several bioactive components when examined by thin layer chromatography coupled with direct bioautography as a screening method. The newly developed infusion overpressured layer chromatographic separation method coupled with direct bioautography assured that only the active components were isolated by means of overrun overpressured layer chromatography with online detection and fractionation. Each of the 5 collected fractions represented one of the five antimicrobial essential oil components designated at the screening. The purity and the activity of the fractions were confirmed with chromatography coupled various detection methods (UV, vanillin-sulphuric acid reagent, direct bioautography). The antibacterial components were identified with GC-MS as thymol, carvacrol, linalool, diethylphthalate, and alpha-terpineol. The oil component diethyl-phthalate is an artificial compound, used as plasticizer or detergent bases in the industry. Our results support that exploiting its flexibility and the possible hyphenations, overpressured layer chromatography is especially attractive for isolation of antimicrobial components from various matrixes

Recent developments in the rapid analysis of plants and tracking their bioactive constituents

Natural products chemistry has witnessed many new developments in the last 5 years like extractions with subcritical water and ionic liquids, LC/HRMS and LC/SPE/cryo-NMR, UHPLC, TLC/MS, MS-based preparative HPLC, comprehensive chromatography (GC × GC, LC × LC), high-throughput screening, introduction of monolithic columns, miniaturisation, and automated structure identification. Nevertheless identifying bioactive constituents in complex plant extracts remains a tedious process. The classical approach of bioassay guided fractionation is time-consuming while off-line screening of extracts does not provide information on individual compounds and sometimes suffers from false positives or negatives. One way out of this is by coupling chromatography with chemical or biochemical assays, so called high resolution screening. An example is the development of HPLC on-line assays for antioxidants. By the post-column addition of a relatively stable coloured radical like DPPH¿ or ABTS¿+, radical scavengers are detected as negative peaks because in a reaction coil they reduce the model radical to its reduced, non-coloured form. When combined with LC/DAD/MS and LC/SPE/NMR, reliable identification of active constituents becomes possible without the necessity of ever isolating them in a classical sense. Also for finding leads for new drugs, combining HPLC with biochemical assays is interesting but technically more difficult. Most enzymes do not work at the organic modifier concentrations commonly encountered in RP-HPLC and the reaction time is often longer requiring dilution and lengthy coils respectively. Therefore, new techniques have to be implemented to gain the required sensitivity for on-line enzyme assays. For stable analytes, high temperature LC offers a solution to the organic modifier problem. When enzymes are highly expensive, like those used in the screening for Cytochrome P450 inhibitors, miniaturisation to chip format may offer a way out. Microreactors (chips) are not only useful for miniaturising larger assays but also offer completely new prospects in phytochemical analysis. One such application is in the sample clean-up of acids and bases like alkaloids. In a lay-out of three parallel channels of 100 ¿m width with the middle one containing organic phase and the two outer ones water of high pH (feed phase) and low pH (trapping phase) such a chip replaces two classical LLE steps but is much faster and requires less solvents and less manpower input

Overpressured layer chromatography: from the pressurized ultramicro chamber to BioArena system

The pressurized ultramicro (UM) chamber as a closed adsorbent layer chamber enables the use of a special chromatoplate and a pump to increase and optimize the mobile phase flow velocity through an optional development distance in an adsorbent layer. This chamber is the basic instrument of overpressured-layer chromatography (OPLC), which is a separation technique that combines the advantages of conventional TLC/HPTLC with those of HPLC. The versions of OPLC instrument, the character and achievement of off-line and on-line OPLC systems in analytical and preparative use are described. The development of BioArena as a complex bioautographic system means an exploitation of the unique advantages of planar-layer system for detection, isolation and identification of new antimicrobials, antineoplastics, biopesticides and other biologically active substances as well as for studying fundamental biochemical reactions and mechanisms

Identification of a new strain of Actinomadura isolated from Saharan soil and partial characterization of its antifungal compounds

One promising strain Actinomadura sp. AC170, isolated from Algerian Saharan soil, with strong antifungal activity against pathogenic and toxinogenic fungi, was selected for further studies. The 16S rRNA results showed a distinct phylogenetic lineage from the other species within the Actinomadura genus. The production of antibiotic substances was investigated using GYEA solid medium. The butanolic extract contained four bioactive spots detected on thin layer chromatography plates. Among these antibiotics, a complex called 170A, which showed the more interesting antifungal activity, was selected and purified by reverse-phase HPLC. This complex is composed of four compounds. Ultraviolet-visible, infrared, mass and H nuclear magnetic resonance spectroscopy studies showed that these molecules contain an aromatic ring substituted by aliphatic chains. These compounds differ from the known antibiotics produced by Actinomadura species

The Analysis of Antimicrobial Testing Vincetoxicum stocksii and Isolation of a Highly Active Compound Against Candida albicans by Using Various Different Techniques.

The purpose is to isolate a natural compound, which shows a high activity against Candida albicans, from plant, Vincetoxicum stocksii. Bio-Assay, Thin layer Chromatography, Column Chromatography, TLC bio Assay, and other extraction techniques are used in order to isolate the active compound. First, bio assay technique is carried out on the crude gum. Next, several flash chromatography columns are carried out in order to isolate the target compound, which has a Rf value of ~0.53 in 10:1 DCM/methanol solvent mixture. The TLC bioassay technique is also carried out in order to confirm the hypothesis that the target compound is indeed active

Two rapid assays for screening of patulin biodegradation

Artículo sobre distintos ensayos para comprobar la biodegradación de la patulinaThe mycotoxin patulin is produced by the blue mould pathogen Penicillium expansum in rotting apples during postharvest storage. Patulin is toxic to a wide range of organisms, including humans, animals, fungi and bacteria. Wash water from apple packing and processing houses often harbours patulin and fungal spores, which can contaminate the environment. Ubiquitous epiphytic yeasts, such as Rhodosporidium kratochvilovae strain LS11 which is a biocontrol agent of P. expansum in apples, have the capacity to resist the toxicity of patulin and to biodegrade it. Two non-toxic products are formed. One is desoxypatulinic acid. The aim of the work was to develop rapid, high-throughput bioassays for monitoring patulin degradation in multiple samples. Escherichia coli was highly sensitive to patulin, but insensitive to desoxypatulinic acid. This was utilized to develop a detection test for patulin, replacing time-consuming thin layer chromatography or high-performance liquid chromatography. Two assays for patulin degradation were developed, one in liquid medium and the other in semi-solid medium. Both assays allow the contemporary screening of a large number of samples. The liquid medium assay utilizes 96-well microtiter plates and was optimized for using a minimum of patulin. The semisolid medium assay has the added advantage of slowing down the biodegradation, which allows the study and isolation of transient degradation products. The two assays are complementary and have several areas of utilization, from screening a bank of microorganisms for biodegradation ability to the study of biodegradation pathways

Effect of bioactive compounds from Sainfoin (Onobrychis viciifolia Scop.) on the in vitro larval migration of Haemonchus contortus: role of tannins and flavonol glycosides

Anthelmintic bioactivity against gastrointestinal nematodes has been associated with leguminous forages supporting the hypothesis of a role of condensed tannins. However, the possibility that other compounds might also been involved has received less consideration. Using bio-guided fractionation, the current study aimed at characterising the biochemical nature of the active compounds present in sainfoin (Onobrychis viciifolia), previously identified as an anthelmintic leguminous forage. The effects of sainfoin extracts were evaluated on 3rd stage larvae (L3) of Haemonchus contortus by using a larval migration inhibition (LMI) assay. Comparison of extracts obtained with several solvent systems showed that the bioactivity was associated with the 70:30 acetone/water extract. Further fractionation of the later allowed the separation of phenolic compounds. By use of a dialysis method, compounds were separated with a molecular weight cut-off of 2000 Da. The in vitro anthelmintic effects of the fraction with condensed tannins was confirmed. In the fraction containing molecules of MW < 2000 Da, 3 flavonol glycosides were identified as rutin, nicotiflorin and narcissin. At 1200 μg/ml, each inhibited significantly migration of larvae. Addition of polyvinyl pyrrolidone (PVPP) to both fractions before incubation restore larval migration. These results confirmed the role of both tannins and flavonol glycosides in the anthelmintic properties of sainfoin

Development of strategies for the prioritization of organic trace substances in water by effect-directed analysis

The protection of the aquatic environment and the supply of clean drinking water to people all over the world are central challenges of our time. Monitoring of the aquatic environment and the input of anthropogenic trace substances into it is therefore very important. However, since aquatic environmental samples often consist of complex substance mixtures, their characterization and evaluation is very demanding. By using generic target analysis methods, selected known anthropogenic trace substances can be detected and quantified very sensitively. For the detection of previously unknown substances, non-target analysis methods have been increasingly used in recent years. However, these methods do not provide information on the relevance of the anthropogenic trace substances occurring in water. In this context, especially all those trace substances are regarded as relevant from which a harmful effect on humans or water organisms is to be expected. For the detection of such effective substances, effect-directed analysis (EDA) can be used. In EDA, a bioassay is combined with a fractionation method and subsequent chemical analysis, the aim being to identify the bioactive substance. The separation method used in this work is high-performance thin-layer chromatography (HPTLC). After chromatography, the bioassay is performed directly on the HPTLC plate. If an effective zone appears in the bioassay, a prioritization strategy is used to clarify the identity of the substance. Due to the complex aquatic samples, a large number of different substances in a zone must still be expected despite the applied HPTLC separation, which makes it difficult to identify the effective substance. Therefore, a strategy to simplify the identification of effective substances should be developed. The aim was to reduce the complexity by multidimensional separation in such a way that chemical analysis can be used to prioritize to a few candidates in the effective fraction. In the first part of the work, a selective two-dimensional HPTLC separation was developed to reduce the number of substances in a bioactive zone. After the first separation dimension (1D) the acetylcholinesterase inhibition assay (AChE assay) was performed and afterwards only the effective zones were extracted from the HPTLC plate. The selected effective zones were separated in a second separation dimension (2D) and the bioassay was performed again. With this 2D separation, the peak capacity could be increased by a factor of 7 compared to a 1D HPTLC gradient development. If real water samples are examined for their effects, an additional structural elucidation must be carried out to clearly identify the unknown bioactive substances. In this work, the developed 2D EDA was therefore connected to a high-performance liquid chromatography (HPLC) with high-resolution mass spectrometry (HRMS) and a non-target screening (NTS) was performed. Using three water samples(drinking water, surface water and purified sewage water) spiked with six effective substances, it was shown that the developed strategy is suitable for the identification of effective substances and that these can be recovered despite repeated extraction. When applying the developed methodology to real samples, it was also possible to assign and quantify the detected effect in several waters to the substance lumichrome and to linear alkylbenzene sulfonates. Genotoxicity is a crucial endpoint for the effect assessment of water samples. However, this endpoint with metabolic activation cannot yet be performed directly on the HPTLC plate. Since many of the genotoxic substances have an indirect genotoxic effect, i.e. they only acquire their activity after metabolic activation; this endpoint was investigated in the present work with the umu assay in the microtiter plate. However, separation with HPTLC, subsequent extraction of effective zones and non-target analysis of the extracts, should also be performed for this assay. Therefore the umu assay in the microtiter plate was integrated into the existing EDA-with-HPTLC concept. In laboratory experiments, sodium hypochlorite was added to the drug metformin in order to simulate the behavior of the substance during water treatment (chlorination). The metformin sample treated with hypochlorite was examined with the umu assay and a genotoxic effect was detected. After HPTLC separation of the chlorinated sample, zones were extracted over the entire retardation range. When the extracted zones were examined with the umu assay, the genotoxic effect could be clearly assigned to one fraction. Using high-resolution mass spectrometry, the genotoxic effect could be assigned to an already known transformation product of metformin. The HPTLC separation and extraction of the zones from the plate led to a reduction of the possible effective candidate masses by a factor of 10 and thus to a clear prioritization in HRMS analysis.Der Schutz der aquatischen Umwelt und die Versorgung der Menschen auf der ganzen Welt mit sauberem Trinkwasser sind zentrale Herausforderungen unserer Zeit. Daher ist die Überwachung der aquatischen Umwelt und des Eintrags von anthropogenen Spurenstoffen in diese von wichtiger Bedeutung. Durch den Einsatz klassischer Target-Analysemethoden können ausgewählte, bekannte anthropogene Spurenstoffe sehr empfindlich detektiert werden. Für die Detektion bislang unbekannter Substanzen werden Non-Target-Analysemethoden eingesetzt. Allerdings liefern diese Methoden keine Aussagen zur Relevanz der im Wasser vorkommenden anthropogenen Spurenstoffe. Als relevant gelten in diesem Zusammenhang all jene Spurenstoffe, von denen eine schädliche Wirkung auf den Menschen oder im Wasser lebende Organismen zu erwarten ist. Zur Detektion wirkender Substanzen kann die Wirkungsbezogene Analytik (WBA) verwendet werden. Bei der WBA wird ein Bioassay mit einer Fraktionierungsmethode und anschließender chemischer Analyse kombiniert, wobei das Ziel die Identifizierung der bioaktiven Substanz ist. Als Fraktionierungsmethode wurde in dieser Arbeit die Hochleistungsdünnschichtchromatographie (HPTLC) angewandt. Nach der Chromatographie wird der Bioassay direkt auf der HPTLC-Platte durchgeführt. Tritt im Bioassay eine wirkende Zone auf, so soll mithilfe einer Priorisierungsstrategie die Identität der Substanz aufgeklärt werden. Aufgrund der komplexen aquatischen Proben muss trotz der angewandten HPTLC-Trennung in einer wirkenden Zone noch immer mit einer Vielzahl verschiedener Substanzen gerechnet werden, die eine Identifizierung der wirkenden Substanz erschweren. Es sollte aus diesem Grund eine Strategie zur Vereinfachung der Identifizierung von wirkenden Substanzen entwickelt werden. Ziel war es, die Komplexität durch mehrdimensionale Trennung so zu verringern, dass mithilfe der chemischen Analyse auf wenige Kandidaten in der wirkenden Zone priorisiert werden kann. Im ersten Teil der Arbeit wurde zur Reduktion der Anzahl von Substanzen in einer bioaktiven Zone eine selektive zweidimensionale HPTLC-Trennung entwickelt. Nach der ersten Trenndimension (1D) wurde der Acetylcholinesterase-Hemmtest (AChE-Assay) durchgeführt und anschließend die wirkenden Zonen von der HPTLC-Platte extrahiert. Die ausgewählten wirkenden Zonen wurden in einer zweiten Trenndimension (2D) nochmals aufgetrennt und abermals der Bioassay durchgeführt. Die Peakkapazität konnte durch diese 2D-Trennung im Vergleich zu einer 1D-HPTLC Gradientenentwicklung um den Faktor 7 gesteigert werden. Wenn reale Wasserproben wirkungsbezogenen untersucht werden, muss für eine eindeutige Identifizierung der unbekannten bioaktiven Substanzen zusätzlich eine Strukturaufklärung durchgeführt werden. In dieser Arbeit wurde deshalb die entwickelte 2D-WBA an eine Hochleistungsflüssigkeitschromatographie (HPLC) mit hochauflösender Massenspektrometrie (HRMS) angebunden und ein Non-Target-Screening (NTS) durchgeführt. Am Beispiel dreier mit sechs wirkenden Substanzen dotierter Wasserproben konnte gezeigt werden, dass sich die entwickelte Strategie für die Identifizierung wirkender Substanzen eignet und dass diese trotz mehrmaliger Extraktion wiedergefunden werden können. Bei der Anwendung der entwickelten Methodik auf Realproben gelang es, in mehreren Wässern die detektierte Wirkung der Substanz Lumichrome sowie linearen Alkylbenzolsulfonaten zuzuordnen. Für die Beurteilung der Wirkungen von Wasserproben ist die Gentoxizität ein entscheidender Endpunkt. Allerdings kann dieser Endpunkt mit metabolischer Aktivierung bislang nicht direkt auf der HPTLC-Platte durchgeführt werden. Da jedoch viele der gentoxischen Substanzen indirekt gentoxisch wirken, wurde dieser Endpunkt mit dem umu-Assay in der Mikrotiterplatte untersucht. Jedoch sollten auch für diesen Assay eine Fraktionierung mit der HPTLC, eine anschließende Extraktion wirkender Zonen und Non-Target-Analyse der Extrakte erfolgen. Dazu wurde der umu-Assay in der Mikrotiterplatte in das bestehende WBA-mit-HPTLC-Konzept integriert. Im Laborexperiment wurde das Arzneimittel Metformin mit Natriumhypochlorit versetzt, um damit das Verhalten der Substanz während der Wasseraufbereitung nachzustellen. Die mit Hypochlorit umgesetzte Metforminprobe wurde mit dem umu-Assay untersucht, wobei eine gentoxische Wirkung detektiert werden konnte. Nach der HPTLC-Trennung der gechlorten Probe wurden anschließend Zonen über den gesamten Retardationsbereich extrahiert. Bei der Untersuchung der extrahierten Zonen mit dem umu-Assay konnte der gentoxische Effekt eindeutig einer Fraktion zugeordnet werden. Mittels hochauflösender Massenspektrometrie gelang es, den gentoxischen Effekt einem bereits beschriebenen Transformationsprodukt von Metformin nachzuweisen. Die HPTLC-Trennung und Extraktion der Zonen von der Platte führte zu einer Reduktion der möglichen wirkenden Kandidatenmassen um den Faktor 10 und somit zu einer deutlichen Priorisierung bei der HRMS-Analyse

Antimitotic action of cornin as a biologically active polypeptide. I. Biochemical properties of cornin

We succeeded in the extraction of a substance from beef cornea and rabbit muscle, that markedly inhibits mitosis of sea urchin eggs. The substance extracted from beef cornea is non-dialysable and it can be separated into three fractions by DEAE-cellulose column. Although everyone of these fractions has an antimitotic action, that of fractions II and III is especially marked. These fractions are one of nucleoproteins that have adenine as base. The substance extracted from rabbit muscle is dialysable, and when it is fractionated through DEAE-cellulose column into three fractions, fraction I has no antimitotic effect but fractions II and III have it. Fraction II is one of nucleoproteins that have hypoxanthine as base. Carnin obtained from beef cornea or from rabbit muscle shows a typical protein wave, but after being treated with gas by passing oxygen through cornin solution the wave height is lowered. Carnin, however, is a very stable substance when kept dry in a desiccator.</p