Natural Product Mixtures Research Articles

Chiral Monoterpenoids in Plants — Enantioselective Chromatographic Analysis, and Their Bioactivity

AbstractFor Abstract see ChemInform Abstract in Full Text.

Multi-component metabolic classification of commercial feverfew preparations via high-field 1H-NMR spectroscopy and chemometrics.

There is increasing interest in evaluating the clinical efficacy of herbal medicines. However, there are significant analytical problems associated with quality control and the measurement of the overall composition of such complex, multi-component mixtures as normally required in the pharmaceutical industry. Here we describe a novel NMR spectroscopic and pattern recognition analytical approach to investigate composition and variability of a commonly used herbal medicine. 600 MHz (1)H-NMR spectroscopy and principal components analysis (PCA) was used to discriminate between batches of 14 commercially available feverfew samples based on multi-component metabolite profiles. Two of the batches were significantly different from the other twelve. The twelve remaining classes could be classified into discrete groups by PCA on the basis of minor differences in overall chemical composition. NMR based pattern recognition (PR) analysis of extracts proved to be superior to PR analysis of HPLC traces of the same mixtures. This work indicates the potential value of NMR combined with PCA for the characterisation of complex natural product mixtures, and the discrimination of samples containing allegedly identical ingredients.

Analysis and screening of combinatorial libraries using mass spectrometry.

Mass spectrometry is a highly selective and high throughput analytical technique that is ideally suited for the identification and purity determination of large numbers of compounds prepared using combinatorial chemistry or for the dereplication of natural products. Compounds may be characterized based on molecular weight, elemental composition and structural features based on fragmentation patterns. When coupled to a separation technique such as high-performance liquid chromatography (HPLC) or capillary electrophoresis, mass spectrometric applications may be expanded to include analysis of complex mixtures. However, the slower speed of the separation step reduces the throughput of the analysis. This review concerns the application of mass spectrometry to the characterization of combinatorial libraries and the screening of library and natural product mixtures. Strategies to enhance the throughput of LC-MS are discussed including fast HPLC and parallel LC-MS. Also, mass spectrometry-based screening methods are described including frontal affinity chromatography-mass spectrometry, gel permeation chromatography LC-MS, direct electrospray mass spectrometry of receptor-ligand complexes, affinity chromatography-mass spectrometry, and pulsed ultrafiltration mass spectrometry.

A new method for separation of 131I, produced by irradiation of natural uranium

Separation of 131I from natural uranium fission product mixtures has been accomplished by sorbing the 131I on special platinum–charcoal sorbent and desorbing by buffer solution (NaHCO3+Na2S2O3). High radiochemical and chemical purity is obtained by this method. Important parameters such as temperature, distillation rate, sorbing and desorbing rates and 131I separation yields have been investigated.

Reversed-phase and size-exclusion chromatography as useful tools in the resolution of peroxidase-mediated (+)-catechin oxidation products

The peroxidase-catalysed oxidation of plant phenolics involves one-electron oxidation reactions, and yields unstable mono-radical species, which couple to generate heterogeneous product mixtures of different degrees of polymerisation. One such phenolic susceptible to oxidation by peroxidase is (+)-catechin. Low-pressure chromatography on Sephadex LH-20, using methanol as mobile phase, resolves the main peroxidase-mediated (+)-catechin oxidation products into a dimeric compound (dehydrodicatechin A) and an oligomeric fraction with a polymerisation degree equal or greater than 5. These pure fractions were used to develop rapid high-performance liquid chromatographic methods, both reversed-phase and size-exclusion chromatography for the direct analysis of the peroxidase-mediated (+)-catechin oxidation products. The joint use of both chromatographic systems permitted the qualitative and quantitative identification of the peroxidase-mediated (+)-catechin oxidation products, and can thus be considered as a useful tool for analysing the complex mixtures of natural bioactive plant products synthesized in reactions catalyzed by plant peroxidases.

Lignoid chemical defenses in the freshwater macrophyte Saururus cernuus

Chemical defense against herbivores has rarely been investigated for freshwater plants, possibly due to the common misconception that herbivory on aquatic macrophytes is low and would not select for chemical defenses. In previous work, the freshwater angiosperm Saururus cernuus was shown to be a low preference food for omnivorous crayfish despite its high nutrient value and relatively soft texture. We used feeding by the crayfish Procambarus clarkii to guide fractionation of the deterrent lipid-soluble extract of this plant, leading to the identification of seven deterrent lignoid metabolites, (–)-licarin A, (+)-saucernetin, (–)-dihydroguaiaretic acid, (–)-sauriols A and B, (–)-saucerneol, and (–)-saucerneol methyl ether. Lignans have been implicated in terrestrial plant chemical defenses as insect growth inhibitors, insect toxins, nematocides, antibacterial, and antifungal agents. However, these activities have rarely been demonstrated using ecologically relevant methodologies in terrestrial systems, and never before in freshwater systems. The widespread nature of lignans amongst very distantly related plants, along with their rich diversity of molecular structure, suggests that they could play a large role in mediating plant-herbivore interactions. In addition to the lignoid compounds we identified, there were other compounds present in low concentration or unstable compounds that were deterrent, that did not appear to be lignans, but that we were unable to identify. This plant thus appears to be defended by a complex mixture of natural products.

Identification and complete spectral assignment for two 4?-methylsterols in an inseparable mixture using 2D NMR

Two acetylated 4α-methylsterols were isolated as an inseparable mixture from the octocoral Psuedoplexaura flagellosa. By the use of concerted 2D NMR (COSY, HSQC, HMBC and T-ROESY) techniques, it was possible to both identify the compounds as the acetates of 4α, 24R-dimethyl-5α-cholest-22-en-3β-ol and 4α, 23, 24R-trimethyl-5α-cholest-22-en-3-β-ol and to assign fully their 1H and 13C spectra. The general usefulness of 2D NMR for identifying individual components of complex mixtures of natural products is discussed. Copyright © 2001 John Wiley & Sons, Ltd.

Quantitative phase analysis of natural products using whole-powder-pattern decomposition

The capability of whole-powder-pattern decomposition in the quantitative phase analysis (QPA) of natural products was investigated using three- to six-component mixtures and pottery bodies. Here, the term pottery body means plastic clay suitable for making pottery and it is compounded of ceramic raw materials. Average errors of the weight fractions for each phase were within 1 weight percent in each mixture of natural products. The amounts of reduced oxides in pottery bodies derived from the X-ray diffraction technique were in good agreement with results obtained by X-ray fluorescence analysis. The present procedure does not require knowledge of crystal structures; it appears adequate for the QPA of natural products.

Ferrocene-based electroactive derivatizing reagents for the rapid selective screening of alcohols and phenols in natural product mixtures using electrospray-tandem mass spectrometry.

The alcohols and phenols of oil of cloves, lemon oil, rose absolut, and oil of peppermint were derivatized with ferroceneoyl azide to generate their ferroceneoyl carbamates. These derivatives are selectively detected at the attomole level, in nanomolar concentrations by electrospray-tandem mass spectrometry (ES-MS/MS) without the need for sample cleanup. The ES-MS/MS analyses of the four essential oils revealed all the expected alcohols, and, in the case of lemon oil, it detected alpha-terpineol as a trace component that was not readily observed by GC-MS. The ES-MS/MS analyses complements the more conventional GC-MS analysis. The ES-MS method has the advantage of speed, selectivity, and sensitivity over GC-MS for detection of a targeted alcohol of a specific mass or structural type. The ES-MS method does not require a chromatographic separation of the components to accomplish its task. In contrast, GC-MS remains the preferred method for the determination of the total constituents of an oil. The ES-MS method may produce artifact ions, especially if the sample is wet and an excess of the ferroceneoyl azide is used; however, the artifacts did not interfere with the analyses.

New diffusion-edited NMR experiments to expedite the dereplication of known compounds from natural product mixtures.

[reaction: see text] We present two new diffusion-edited NMR experiments, improved DECODES and HETDECODES, that sort the constituents in a mixture by their individual diffusion coefficients. These experiments should allow the partial NMR spectral assignment and cursory structure elucidation of compounds in a complex mixture as an aid in the dereplication of known or nuisance compounds.

Hydrophilic Interaction Chromatography−Electrospray Mass Spectrometry Analysis of Polar Compounds for Natural Product Drug Discovery

For the drug discovery efforts currently taking place within the pharmaceutical industry, natural product extracts have been found to provide a valuable source of molecular diversity which is complementary to that provided by traditional synthetic organic methods or combinatorial chemistry. However, there exists a need for analytical tools that can facilitate the separation and characterization of components from these sources in a rapid manner. Specifically, the evaluation of highly polar compounds (i.e., compounds that cannot be retained on traditional reversed-phase stationary phases) has been challenging, and a hydrophilic interaction chromatography-electrospray ionization mass spectrometry (HILIC-ESI-MS) method was developed to meet this need. In this investigation, amide-, Polyhydroxyethyl Aspartamide-, and cyclodextrin-based packings provided superior performance for the analysis of a set of polar natural product compounds. The properties of the mobile-phase buffers also greatly impacted the separations, and relative to other volatile buffering agents, ammonium acetate at a concentration of approximately 6.5 mM was determined to facilitate optimal HILIC retention, reproducibility, and durability. An optimized HILIC-ESI-MS system was successfully applied for the analysis of complex natural product mixtures. The techniques described in this report should also prove useful for the analysis of polar compounds from synthetic sources of molecular diversity such as combinatorial chemistry.

Application of High-Speed Countercurrent Chromatography/Electrospray Ionization Mass Spectrometry (HSCCC/ESIMS) in Natural Products Chemistry

In this paper, we describe the technique of analytical highspeed countercurrent chromatography coupled with electrospray ionization mass spectrometry (HSCCC/ESIMS) for the first time, and evaluate its performance in terms of chromatography and mass spectrometry. This technique demonstrates the capability and resolving power of HSCCC in the separation of a few natural product mixtures and utilizes ESIMS as a detector with continuous detection and high sensitivity. Application of this HSCCC/ESIMS system is illustrated by the separation and detection of biologically important compounds.

Approaches to Automating the Dereplication of Bioactive Natural Products—The Key Step in High Throughput Screening of Bioactive Materials From Natural Sources

The rapid identification of the bioactive component(s) of natural product mixtures in high throughput screening programs has become a critical factor to ensure that this source of diverse chemotypes can compete effectively with chemical compound libraries and combinatorial synthetic efforts. The effective use of automated procedures and databases in the isolation, identification and biological profiling of bioactive compounds will be described. In addition, the potential of new technologies to enhance this process will be discussed as well as the possible reintroduction of TLC as a parallel dereplication method.

Development of non-destructive methods for the determination of airborne pollutants in pine needles: identification of trace constituents in radiata pine epicuticular wax

In an attempt to develop non-destructive work-up procedures for monitoring lipophilic pollutants in the air by analysis of pine needle epicuticular wax, a project was undertaken to identify substances that interfere with the normal gas-chromatographic quantitations. Epicuticular wax was extracted from needles of radiata pine and processed to remove the bulk estolides and resin and fatty acids. Fractionation of the residue on a silica gel column gave three fractions, representing less than 0.2% of the wax. The fractions contained a mixture of natural products and anthropogenic chemicals. Among the natural products were sesquiterpenes, comprising mainly amorphene, cadinenes, germacrene and cadalene; bisnor-, nor- and diterpene hydrocarbons; methyl esters of resin acids; and 9,10-secodehydroabietanes of unknown ring-A structure. Some of these compounds have not been reported from pine needle epicuticular wax previously. Anthropogenic chemicals identified include polycyclic aromatic hydrocarbons. The organochlorine compounds of environmental importance were hexachloro-cyclohexanes (including lindane), hexachlorobenzene, pentachloroanisole, chlordanes, dieldrin, p,p′-DDE, p,p′-DDT, and penta-, hexa- and heptachlorobiphenyls. The organochlorine compounds were present at concentration orders of magnitude lower than the interfering compounds, and therefore had to be identified using gas chromatography-mass spectrometry methods. By identifying the interfering compounds, non-destructive work-up procedures could be developed in order to retain the integrity of all of the anthropogenic compounds contributing to environmental pollution.

Preparative liquid chromatography with analytical separation quality : Internal injection/displacement reversed-phase high-performance liquid chromatography

A method for the enrichment and separation of compounds, called interval injection/displacement reversed-phase high-performance liquid chromatography (HPLC), was applied to several analytical and preparative separations. In principle, the surface of a reversed-phase stationary phase, equilibrated with a weak mobile phase, is “coated” with compounds by stepwise injection of small samples. Distinct time intervals between injections allow the stationary phase to re-equilibrate. In this way, sample enrichment can be achieved in ranges comparable to those in overload elution or displacement HPLC. Sample fractionation proceeds in a similar manner to conventional displacement modes. Also under full mass load conditions, a quality of separation similar to that in analytical elution HPLC is achieved. The results obtained indicate that a very large number of samples can be applied to conventionally sized columns without overload problems, the full mass capacity of a column can be utilized and the method is useful for enriching and separating compounds with a wide range of polarities. Interval injection/displacement reversed-phase HPLC was successfully applied to complex mixtures of natural products and enzyme assay mixtures. Further, this technique is likely to be useful for the analysis of trace compounds and quality control of chemicals. The experiments reported were performed on highly unstable natural products (thiophenic and benzofuran compounds) from Tagetes plants.

Acaricidal activity of kupetaba, a ground mixture of natural products, against Rhipicephalus appendiculatus

Eight substances were screened for the purpose of selecting a natural product as an acaricide for use within an integrated tick management system. One substance proved to be effective as an acaricide against all stages of Rhipicephalus appendiculatus and was named ‘kupetaba’. It is a ground mixture of dried tobacco leaves (family Solanaceae) and a mineral called ‘Magani soda’ which is mined around Lake Magadi in the Rift Valley province of Kenya. Investigation showed that kupetaba is commonly sold in local markets in East, West and Central Africa, where it is used for many purposes. The substance prevented the completion of all feeding phases of the tick, suppressed the oviposition capacity of the engorged ticks and drastically reduced the hatchability of the eggs. Larvae and nymphs were killed within 24 h of the application of the substance on calves' ears, while large numbers of adult ticks were also killed within 2–3 days of application during in vitro experiments. The suitability of the natural product as a local acaricide which resource-poor farmers in Africa can use to replace commercial acaricides within an integrated tick management system is discussed.

Centrifugal partition chromatography in the separation of natural products

AbstractRecent innovations in liquid/liquid chromatographic techniques have led to a resurgence of interest in their use for the separation of natural products. Commercially available instruments which rely on centrifugal acceleration to speed up separation times are based either on a battery of cartridges or on a rotating multilayer coil and can be employed either as analytical tools or for preparative purposes. While solvent consumption is low, milligram to gram quantities are capable of being separated. Aqueous and non‐aqueous two‐phase solvent systems are compatible with these techniques and numerous applications involving simple and complex mixtures of natural products, both polar and non‐polar, have been demonstrated.

New procedure for direct bioautographic TLC assay as applied to a tincture of Ranunculus bulbosus

A new procedure for TLC bioautographic assay using a flooding method to inoculate the developed plates is described. Details about several microbiological parameters allow good reproducibility for this method which appears rapid, easy to perform and requiring no specialized equipment. Significantly results obtained with a commercial tincture of Ranunculus bulbosus illustrate this method which can be extented to complex mixtures of natural products in order to isolate active fractions.

Applications of Capillary Supercritical Fluid Chromatography-Supercritical Fluid Extraction to Natural Products

Capillary supercritical fluid chromatography (SFC) is uniquely suited for the separation of complex mixtures of natural products having nonpolar to moderately polar components with a molecular weight range of 100–1000 Daltons. This study describes several generic separation problems where SFC, or SFC coupled with micro-supercritical fluid extraction (SFE), can be utilized to the analyst's advantage. For example, capillary SFC can be used to separate the analytes of Interest from a relatively nonvolatile sample matrix without resorting to sample preparation prior to chromatography. Other applications of capillary SFC include the separation of reaction products from higher molecular weight starting materials and the deformulation of commercial products. Capillary SFC, when coupled with micro-scale SFE also permits the characterization of small samples, such as portions of single seeds and extractables from single, live Insects.

Separation of phenolics (benzoic acids, cinnamic acids, phenylacetic acids, quinic acid esters, benzaldehydes and acetophenones, miscellaneous phenolics) and coumarins by reversed-phase high-performance liquid chromatography

The high-performance liquid chromatographic separation of a relative large series of simple phenolics and related substances by means of a reversed-phase system is described. The system consists of a LiChrosorb RP-18 (10 μm) Knauer column and a combination of isocratic and linear-gradient elution (solvent a, water—formic acid (95:5); solvent B, methanol; temperature, 35°C). The series of compounds studied embraces derivatives of benzoic acid (29 compounds), phenylacetic acid (11 compounds), cinnamic acid (26 compounds) and dihydrocinnamic acid (8 compounds), quinic acid esters of phenolic benzoic and cinnamic acid derivatives (9 compounds), benzaldehydes and acetophenones (25 compounds), miscellaneous phenolics (27 compounds) and coumarins (43 compounds). Amongst these substances, phenolic acids, lactones, aldehydes and ketones are represented. The possible use of the system for the analysis of complex mixtures of natural products as well as for the elucidation of the structures of more complex natural compounds is discussed.