Complex Natural Mixtures Research Articles

Crystalline-Sponge-Based Structural Analysis of Crude Natural Product Extracts.

Characterization of complex natural product mixtures to the absolute structural level of their components often requires significant amounts of starting materials and lengthy purification process, followed by arduous structure elucidation efforts. The crystalline sponge (CS) method has demonstrated utility in the absolute structure elucidation of isolated organic compounds at miniscule quantities compared to conventional methods. In this work, we developed a new CS-based workflow that greatly expedites the in-depth structural analysis of crude natural product extracts. Using a crude extract of the red alga Laurencia pacifica, we showed that CS affinity screening prior to compound isolation enables prioritization of analytes present in the extract, and we subsequently resolved the molecular structures of six sesquiterpenes with stereochemical clarity from around 10 mg crude extract. This study demonstrates a new chemotyping workflow that can greatly accelerate natural product discovery from complex samples.

Essential Oils and Their Single Compounds in Cosmetics—A Critical Review

Essential oils are widely incorporated in cosmetic products, perfumes and related household products due to the variety of their properties but mainly due to their pleasant odour. The composition of these volatile natural complex mixtures may vary depending on the quality of plant material from which they were obtained and the extraction method by which they were derived. These factors are also important in ensuring the safe use of essential oils in personal care products. As they contain compounds with varied chemical structure and effects, skin sensitivity and irritations as well as other symptoms may arise after their application. Although essential oils are considered as safe and nontoxic when used at low concentrations, available scientific literature indicates that essential oils and their compounds may possess a strong allergy potential. This review focuses on side effects and allergy contact dermatitis caused by selected essential oils and their single compounds in cosmetic products, summarizing data from the most recent scientific literature.

Inhibition of Epithelial TNF-α Receptors by Purified Fruit Bromelain Ameliorates Intestinal Inflammation and Barrier Dysfunction in Colitis.

Activation of the TNF-α receptor (TNFR) leads to an inflammatory response, and anti-TNF therapy has been administered to reduce inflammation symptoms and heal mucosal ulcers in inflammatory bowel disease (IBD). Bromelain, a complex natural mixture of proteolytic enzymes, has been shown to exert anti-inflammatory effects. This study aimed to investigate the effect of purified fruit bromelain (PFB)-induced inhibition of epithelial TNFR in a rat colitis model. Colitis was established by intracolonic administration of 2, 4, 6-trinitrobenzene sulfonic acid. Expression of TNFR1 and TNFR2 was measured by quantitative RT-PCR and western blotting. The effect of PFB on colitis was evaluated by examining the inflammatory response and intestinal epithelial barrier function. Our results showed that both TNFR1 and TNFR2 expression were significantly increased in a colitis model, and the increase was significantly reversed by PFB. Colitis symptoms, including infiltration of inflammatory cells, cytokine profiles, epithelial cell apoptosis, and epithelial tight junction barrier dysfunction were significantly ameliorated by PFB. Compared with fruit bromelain and stem bromelain complex, the inhibition of TNFR2 induced by PFB was stronger than that exhibited on TNFR1. These results indicate that PFB showed a stronger selective inhibitory effect on TNFR2 than TNFR1. In other words, purification of fruit bromelain increases its selectivity on TNFR2 inhibition. High expression of epithelial TNFRs in colitis was significantly counteracted by PFB, and PFB-induced TNFR inhibition ameliorated colitis symptoms. These results supply novel insights into potential IBD treatment by PFB.

New synthetic lipid antigens for rapid serological diagnosis of tuberculosis.

BackgroundDuring pulmonary tuberculosis (PTB) antibodies are generated to trehalose esters of mycolic acids which are cell wall lipids of Mycobacterium tuberculosis (Mtb). Attempts have been made to use these complex natural mixtures in serological tests for PTB diagnosis.AimThe aim of this work was to determine whether a serological test based on a panel of defined individual trehalose esters of characteristic synthetic mycolic acids has improved diagnostic accuracy in distinguishing patients with culture positive PTB from individuals who were Mtb culture negative.MethodOne hundred serum samples from well-characterized patients with presumptive tuberculosis, and diagnosed as having pulmonary smear and culture positive TB, or being culture and smear negative were evaluated by ELISA using different combinations of synthetic antigens and secondary antibodies. Using cut-off values determined from these samples, we validated this study blind in samples from a further 249 presumptive TB patients.ResultsWith the first 100 samples, detailed responses depended both on the precise structure of the antigen and on the secondary antibody. Using a single antigen, a sensitivity/specificity combination for smear and culture positive PTB detection of 85 and 88% respectively was achieved; this increased to 96% and 95% respectively by a statistical combination of the results with seven antigens. In the blind study a sensitivity/specificity of 87% and 83% was reached with a single antigen. With some synthetic antigens, the responses from all 349 samples were significantly better than those with the natural mixture. Combining the results for seven antigens allowed a distinction between culture positive and negative with a ROC AUC of 0.95.ConclusionWe have identified promising antigen candidates for serological assays that could be used to diagnose PTB and which could be the basis of a much-needed, simple, rapid diagnostic test that would bring care closer to communities.

Investigation of the ozonation products of natural complex mixtures using Fourier transform ion cyclotron resonance mass spectrometry.

Natural complex mixtures such as oil and dissolved organic matter play an important role in the economy and in the global carbon cycle. One of the most promising approaches for the investigation of the chemical structure of such substances is the combination of the high-resolution mass spectrometry and selective chemical reactions. Here, we report the investigation of the ozonation products of natural complex mixtures using Fourier transform ion cyclotron resonance mass spectrometry. Ozonation of crude oil results in the appearance of the new compounds with high content (up to 9 atom) of oxygen. Isotopic exchange reaction showed that those oxygen stem from the carbonyl groups. Ozonation of the dissolved organic matter leads to the destruction of the substance and shift towards the region of the saturated compounds.

Magnetic nanoparticles and high-speed countercurrent chromatography coupled in-line and using the same solvent system for separation of quercetin-3-O-rutinoside, luteoloside and astragalin from a Mikania micrantha extract

A new in-line method of magnetic nanoparticles (MNPs) coupled with high-speed countercurrent chromatography (HSCCC) using a same solvent system during the whole separation process was established to achieve the rapid separation of flavonoids from Mikania micrantha. The adsorption and desorption capacities of five different MNPs for flavonoid standards and Mikania micrantha crude extract were compared and the most suitable magnetic nanoparticle Fe3O4@SiO2@DIH@EMIMLpro was selected as the in-line MNP column. An in-line separation system was established by combining this MNP column with HSCCC through a six-way valve. The comparison between two solvent systems n-hexane-ethyl acetate-methanol-water (3:5:3:5, v/v) and ethyl acetate-methanol-water (25:1:25, v/v) showed that the latter solvent system was more suitable for simultaneously in-line separating three flavonoids quercetin-3-O-rutinoside, luteoloside and astragalin from Mikania micrantha. The purities of these three compounds with the ethyl acetate-methanol-water solvent system were 95.13%, 98.54% and 98.19% respectively. Results showed the established in-line separation system of MNP-HSCCC was efficient, recyclable and served to isolate potential flavonoids with similar polarities from natural complex mixtures. The in-line combination of magnetic nanoparticles with high-speed countercurrent chromatography eluting with the same solvent system during the whole separation process was established for the first time.

Screening inhibitors of xanthine oxidase from natural products using enzyme immobilized magnetic beads by high-performance liquid chromatography coupled with tandem mass spectrometry.

In this study, high-performance liquid chromatography coupled with tandem mass spectrometry was used to assess the results of bioactive compound screening from natural products using immobilized enzyme magnetic beads. We compared three commercial magnetic beads with modified amino, carboxy, and N-hydroxysuccinimide groups, respectively. Amino magnetic beads performed best for immobilization and were selected for further experiments. Xanthine oxidase was immobilized on amino magnetic beads and applied to screen potential inhibitors in fresh Zingiber officinale Roscoe, extracts of Scutellaria baicalensis Georgi, and Pueraria lobata Ohwi. In total, 12 potential xanthine oxidase ligands were identified from fresh Zingiber root and Scutellaria root extracts, of which eight were characterized and the concentration required for 50% inhibition was determined. Preliminary structure-function relationships were discussed based on these results. A convenient and effective method was therefore developed for the identification of active compounds from complex natural product mixtures.

Analysis of terpenes and turpentines using gas chromatography with vacuum ultraviolet detection.

The separation and identification of natural mixtures of terpenes is challenging and laborious. A gas chromatographic method based on vacuum ultraviolet spectroscopic detection, which is characterized by full-scan absorption in the range of 125-240 nm, was developed and applied to analyze terpenes. In this study, the vacuum ultraviolet absorption spectra of 41 different standard terpenes were investigated and compared. The spectra were found to be highly featured and easily differentiated. Several commercial turpentine samples were analyzed and the vacuum ultraviolet detector demonstrated good specificity for qualitative identification of constituent terpenes. A total of 31 terpenes were detected in the four turpentine samples. α-Pinene was the predominant terpene ranging from 744.2 ± 9.7 to 917 ± 21 mg/mL. The other major constituents in the turpentines included β-pinene, δ-3-carene, camphene, and p-isopropyltoluene. Deconvolution of co-eluting signals of terpenes was achieved utilizing the data analysis software. The technique has been demonstrated to be a powerful tool for reliable and accurate qualitative and quantitative analysis of terpenes from complex natural mixtures.

Analysis of Pandanus Extracts by Reversed Phase High Performance Liquid Chromatography

The Pandanus species (Pandanaceae family) was investigated, in order to set up a library of their chromatographic profiles. From a literature review, the most common Pandanus plant that was greatly examined was P. amaryllifolius (the fragrant screw pine), followed by P. sanderi. Here, the chromatographic data from an extract of a relatively unfamiliar Pandanus is also presented. The leaves of P. monotheca were extracted by using methanol. Later, the extract was filtered, prior to a Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) experiments. The automated system was set to run by gradient elution (acetonitrile:water). The flow rate was maintained at 1 ml/min, otherwise stated in a number of attempts. The ultra-violet absorbance of the Pandanus’ constituents was measured via Multiple Wavelength Detector. From the chromatogram, the major peaks for Pandanus’ compounds were eluted (λ = 220, 260 and 360 nm) and the retention times were recorded. The lower absorbance readings of other minor peaks could be due to various minor secondary metabolites, such as the furolignan. The different elution time depends on the polarity of the composition. In short, Pandanus’ phytochemicals could be obtained via alcoholic extraction. The utilisation of modern chromatographic applications enables the scientists to discover and subsequently, purify single component of the complex natural mixtures. This finding can be beneficial to the research areas in botanical study, pharmacognostical assessment and biotechnology industry. It is aimed that more Pandanus species could be learnt for the future.

Analysis of Pandanus Extracts by Reversed Phase High Performance Liquid Chromatography

The Pandanus species (Pandanaceae family) was investigated, in order to set up a library of their chromatographic profiles. From a literature review, the most common Pandanus plant that was greatly examined was P. amaryllifolius (the fragrant screw pine), followed by P. sanderi. Here, the chromatographic data from an extract of a relatively unfamiliar Pandanus is also presented. The leaves of P. monotheca were extracted by using methanol. Later, the extract was filtered, prior to a Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) experiments. The automated system was set to run by gradient elution (acetonitrile:water). The flow rate was maintained at 1 ml/min, otherwise stated in a number of attempts. The ultra-violet absorbance of the Pandanus’ constituents was measured via Multiple Wavelength Detector. From the chromatogram, the major peaks for Pandanus’ compounds were eluted (λ = 220, 260 and 360 nm) and the retention times were recorded. The lower absorbance readings of other minor peaks could be due to various minor secondary metabolites, such as the furolignan. The different elution time depends on the polarity of the composition. In short, Pandanus’ phytochemicals could be obtained via alcoholic extraction. The utilisation of modern chromatographic applications enables the scientists to discover and subsequently, purify single component of the complex natural mixtures. This finding can be beneficial to the research areas in botanical study, pharmacognostical assessment and biotechnology industry. It is aimed that more Pandanus species could be learnt for the future.

21 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometer Greatly Expands Mass Spectrometry Toolbox.

We provide the initial performance evaluation of a 21 Tesla Fourier transform ion cyclotron resonance mass spectrometer operating at the Environmental Molecular Sciences Laboratory at the Pacific Northwest National Laboratory. The spectrometer constructed for the 21T system employs a commercial dual linear ion trap mass spectrometer coupled to a FTICR spectrometer designed and built in-house. Performance gains from moving to higher magnetic field strength are exemplified by the measurement of peptide isotopic fine structure, complex natural organic matter mixtures, and large proteins. Accurate determination of isotopic fine structure was demonstrated for doubly charged Substance P with minimal spectral averaging, and 8158 molecular formulas assigned to Suwannee River Fulvic Acid standard with root-mean-square (RMS) error of 10ppb. We also demonstrated superior performance for intact proteins; namely, broadband isotopic resolution of the entire charge state distribution of apo-transferrin (78kDa) and facile isotopic resolution of monoclonal antibody under a variety of acquisition parameters (e.g., 6s time-domains with absorption mode processing yielded resolution of approximately 1M at m/z = 2700). Graphical Abstract ᅟ.

Profiling and classification of French propolis by combined multivariate data analysis of planar chromatograms and scanning direct analysis in real time mass spectra

Quality control of propolis is challenging, as it is a complex natural mixture of compounds, and thus, very difficult to analyze and standardize. Shown on the example of 30 French propolis samples, a strategy for an improved quality control was demonstrated in which high-performance thin-layer chromatography (HPTLC) fingerprints were evaluated in combination with selected mass signals obtained by desorption-based scanning mass spectrometry (MS). The French propolis sample extracts were separated by a newly developed reversed phase (RP)-HPTLC method. The fingerprints obtained by two different detection modes, i.e. after (1) derivatization and fluorescence detection (FLD) at UV 366nm and (2) scanning direct analysis in real time (DART)-MS, were analyzed by multivariate data analysis. Thus, RP-HPTLC-FLD and RP-HPTLC-DART-MS fingerprints were explored and the best classification was obtained using both methods in combination with pattern recognition techniques, such as principal component analysis. All investigated French propolis samples were divided in two types and characteristic patterns were observed. Phenolic compounds such as caffeic acid, p-coumaric acid, chrysin, pinobanksin, pinobanksin-3-acetate, galangin, kaempferol, tectochrysin and pinocembrin were identified as characteristic marker compounds of French propolis samples. This study expanded the research on the European poplar type of propolis and confirmed the presence of two botanically different types of propolis, known as the blue and orange types.

Development of an Online Cell-Based Bioactivity Screening Method by Coupling Liquid Chromatography to Flow Cytometry with Parallel Mass Spectrometry.

This study describes a new platform for the fast and efficient functional screening for bioactive compounds in complex natural mixtures using a cell-based assay. The platform combines reversed-phase liquid chromatography (LC) with online flow cytometry (FC) and mass spectrometry (MS). As a model (an example or proof-of-concept study) we have used a functional calcium-flux assay in human neuroblastoma SH-SY5Y cells stably overexpressing the α-7 nicotinic acetylcholine receptor (α7-nAChR), a potential therapeutic target for central nervous system (CNS) related diseases. We have designed the coupled LC-FC system employing the neuroblastoma cells followed by analytical and pharmacological evaluation of the hyphenated setup in agonist and mixed antagonist-agonist assay modes. Using standard receptor ligands we have validated pharmacological responses and standardized good assay quality parameters. The applicability of the screening system was evaluated by analysis of various types of natural samples, such as a tobacco plant extract (in agonist assay mode) and snake venoms (in mixed antagonist-agonist assay mode). The bioactivity responses were correlated directly to the respective accurate masses of the compounds. Using simultaneous functional agonist and antagonist responses nicotine and known neurotoxins were detected from tobacco extract and snake venoms, respectively. Thus, the developed analytical screening technique represents a new tool for rapid measurement of functional cell-based responses and parallel separation and identification of compounds in complex mixtures targeting the α7-nAChR. It is anticipated that other fast-response cell-based assays (e.g., other ion flux assays) can be incorporated in this analytical setup.

Development of a Total Organic Carbon method for the quantitative determination of solubility enhancement by cyclodextrins: Application to essential oils

Formation of inclusion complexes with cyclodextrins (CDs) is known to enhance guest solubility in aqueous medium. Different techniques allow determining the evolution in solubility of individual guest compounds. However, examination of mixtures solubility encapsulated in CDs is still a challenge. This is mainly related to the difference in the response of mixture components to the applied technique or to the fact that most of the conventional methods examine the signal of an individual constituent of the mixture. Thus, applying current techniques may not reflect the behavior of the whole mixture. Here, we used for the first time Total Organic Carbon (TOC) analysis to explore and assess the efficiency of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) to enhance the solubility of natural complex mixtures such as essential oils (EOs). Phase solubility studies were performed for eleven EOs with HP-β-CD. The TOC method has provided good validation parameters for linearity, precision and accuracy. For further validation of the method, phase solubility studies were performed with HP-β-CD for eugenol, as a model EO component. The eugenol solubility was determined by UV–Visible and TOC analyses in order to compare the results. Data obtained from both methods were similar (p < 0.05), thereby proving the effectiveness of the developed TOC method. Finally, the phase solubility diagrams of EOs showed that the solubilizing potential of CD increased proportionally with the decrease in EO intrinsic solubility. Results proved that TOC could be successfully applied to investigate CD/guest inclusion complexes and is expected to have a broad range of applications in the field of mixtures encapsulation.

Silver nanoparticles formation by extracellular polymeric substances (EPS) from electroactive bacteria

Microbial extracellular polymeric substances (EPS) excreted from microorganisms were a complex natural biological polymer mixture of proteins and polysaccharides, which played an important roles in the transport of metals, such as Ag(+). Electroactive bacteria, is an important class of environmental microorganisms, which can use iron or manganese mineral as terminal electron acceptors to generate energy for biosynthesis and cell maintenance. In this work, the EPS extracted of three electroactive bacteria (Shewanella oneidensis, Aeromonas hydrophila, and Pseudomonas putida) were used for reducing Ag(+) and forming silver nanoparticles (AgNPs). Results showed that all the three microbial EPS could reduce Ag(+) to AgNPs. The formed AgNPs were characterized in depth by the UV-visible spectroscopy, X-ray diffraction, X-ray photoelectron spectroscopy, and high-resolution transmission electron microscopy. The main components in the EPS from the three electroactive bacteria were analyzed. The presence of cytochrome c in these EPS was confirmed, and they were found to contribute to the reduction of Ag(+) to AgNPs. The results indicated that the EPS of electroactive bacteria could act as a reductant for AgNPs synthesis and could provide new information to understand the fate of metals and their metal nanoparticles in the natural environments.

Application of diffusion-edited and solvent suppression ¹H-NMR to the direct analysis of markers in valerian-hop liquid herbal products.

The rising trend to consume herbal products for the treatment and/or prevention of minor ailments together with their chemical and pharmacological complexity means there is an urgent need to develop new approaches to their quality and stability. This work looks at the application of one-dimensional diffusion-edited (1)H-NMR spectroscopy (1D DOSY) and (1)H-NMR with suppression of the ethanol and water signals to the characterisation of quality and stability markers in multi-component herbal medicines/food supplements. The experiments were performed with commercial tinctures of Valeriana officinalis L. (valerian), expired and non-expired, as well as its combination with Hummulus lupulus L. (hops), which is one of the most popular blends of relaxant herbs. These techniques did not require purification or evaporation of components for the qualitative analysis of the mixture, but only the addition of D2 O and TSP. The best diagnostic signals were found at δ 7 ppm (H-11, valerenic acid), δ 4.2 ppm (H-1, hydroxyvalerenic acid) and δ 1.5-1.8 ppm (methyl groups in prenylated moieties, α-acids/prenylated flavones). This work concludes on the potential value of 1D DOSY (1)H-NMR to provide additional assurance of quality in complex natural mixtures.

Positively charged and bipolar layered poly(ether imide) nanofiltration membranes for water softening applications

Poly(ether imide) (PEI) ultrafiltration membranes were chemically modified with branched poly(ethyleneimine) to obtain nanofiltration (NF) membrane Cat PEI with a positive charge in the pH range below 9. An oppositely charged polyelectrolyte layer was deposited on the resulting membrane surface by using sodium polystyrene sulfonate (PSSNa) and sodium polyvinyl sulfonate (PVSNa) to prepare a bipolar layered membrane NF Cat PEI_PSS and Cat PEI_PVS having a negatively charged surface and positively charged pores. Cat PEI exhibited good performance to remove multivalent cations (more than 90% of Ca2+) from single salt solutions except in presence of sulfate ions. Adding an anionic polyelectrolyte layer onto the positively charged surface resulted in a significant enhancement of rejection performance even in presence of sulfate anions. Application of the prepared membranes in water softening of natural complex mixtures was successful for the different studied membranes and a large decrease of hardness was obtained. Moreover, Cat PEI_PSS showed a good selectivity for nitrate removal. Fouling experiments were carried out with bovine serum albumin, as model protein foulant. Cat PEI_PSS showed much better fouling resistance than Cat PEI with a quantitative flux recovery ratio.

Contrasted effects of natural complex mixtures of PAHs and metals on oxygen cycle in a microbial mat

The contamination of polluted environments is often due to a complex mixture of pollutants sometimes at trace levels which nevertheless may have significant effects on the diversity and functioning of organisms. The aim of this study was to assess the functional responses of a microbial mat exposed to a natural complex mixture of PAHs and metals as a function of the maturation stage of the biofilm. Microbial mats sampled in a slightly polluted environment were exposed to contaminated water of a retention basin of an oil refinery. The responses of the microbial mats differed according to season. In spring 2012, strong inhibition of both oxygen production and respiration was observed relative to the control, with rates representing less than 5% of the control after 72h of incubation. A decrease of microbial activities was followed by a decrease of the coupling between autotrophs and heterotrophs. In contrast, in autumn 2012, no significant changes for oxygen production and respiration were observed and the coupling between autotrophs and heterotrophs was not altered. The differences observed between the spring and autumn mats might be explained by the maturity of the microbial mat with dominance of heterotrophic bacteria in spring, and diatoms and cyanobacteria in autumn, as well as by the differences in the chemical composition of the complex mixture of PAHs and metals.

Connecting phenotype and chemotype: high-content discovery strategies for natural products research.

In recent years, the field of natural products has seen an explosion in the breadth, resolution, and accuracy of profiling platforms for compound discovery, including many new chemical and biological annotation methods. With these new tools come opportunities to examine extract libraries using systematized profiling approaches that were not previously available to the field and which offer new approaches for the detailed characterization of the chemical and biological attributes of complex natural products mixtures. This review will present a summary of some of these untargeted profiling methods and provide perspective on the future opportunities offered by integrating these tools for novel natural products discovery.

Chemical assay-guided natural product isolation via solid-supported chemodosimetric fluorescent probe.

As a new model of chemical assay-guided natural product isolation, an effective chemodosimetric assay system was devised. Our chemical assay system features a fluorogenic chemodosimeter immobilized on a solid support, which offers advantages in identifying the desired compounds in complex natural product mixtures. To isolate only compounds with the target functional groups, the click chemistry concept was adopted. The model system presented here was developed for natural products with a terminal alkyne. Using our newly designed alkyne sensing beads with the aforementioned features, we have readily identified, quantified, and isolated compounds with a terminal alkyne group from plant extracts.