Analysis of commercial proanthocyanidins. Part 5: A high resolution mass spectrometry investigation of the chemical composition of sulfited wattle (Acacia mearnsii De Wild.) bark extract.

Wattle (Acacia mearnsii De Wild., Leguminosae) bark extract is used commercially to tan leather and manufacture adhesives. The extract is treated with sodium hydrogen sulfite (sulfited) to improve its tanning properties. These include reduced viscosity, improved solubility, and better raw skin penetration. High resolution ESIMS allows unambiguous assignment of sulphur-containing monomeric and oligomeric products from sulfitation. It reveals that during sulfitation the constituent flavan-3-ol building blocks are sulfited at both C-2 and C-4 by a sulfite ion. A sulfonic acid moiety is introduced at C-2 to open the pyran ring, and C-4 to cleave the interflavanyl bond and reduce the degree of polymerization, respectively, explaining the improved tanning properties. MS2 fragmentation spectra and comparison with unsulfited extract support the interpretation of peaks and composition of sufited wattle bark extract. It also supports our published work that mimosa bark extract consists of a catechin or gallocatechin starter unit and fisetinidol or robinetinidol extender units.

Labdane and Clerodane Diterpenoids from Colophospermum mopane.

Five labdane (1-5), an isolabdane (6), and five clerodane diterpenoids (7-11), were isolated from seeds, husks, and leaves of Colophospermum mopane. Compounds 1-3 and 6-9 are new, and their structures were elucidated by means of physical data analysis (1D and 2D NMR, HRESIMS). The absolute configurations of 1, 7, and 10 were determined by single-crystal X-ray diffraction with Cu Kα radiation. For compounds 2 and 6, the absolute configurations were established by the modified Mosher's method and corroborated by comparison of experimental and calculated electronic circular dichroism spectra of their 3-p-bromobenzoate derivatives. The crude extracts and compounds were evaluated for antimicrobial activity. The leaf extract was the most active against Staphylococcus aureus (125 µg/mL). Compound 11 showed the best inhibitory activity, with minimum inhibitory values of 15.6 µg/mL against Escherichia coli and Staphylococcus aureus and 31.3 µg/mL against Enterococcus faecalis.

hERG Channel Inhibitory Daphnane Diterpenoid Orthoesters and Polycephalones A and B with Unprecedented Skeletons from Gnidia polycephala.

The hERG channel is an important antitarget in safety pharmacology. Several drugs have been withdrawn from the market or received severe usage restrictions because of hERG-related cardiotoxicity. In a screening of medicinal plants for hERG channel inhibition using a two-microelectrode voltage clamp assay with Xenopus laevis oocytes, a dichloromethane extract of the roots of Gnidia polycephala reduced the peak tail hERG current by 58.8 ± 13.4% (n = 3) at a concentration of 100 µg/mL. By means of HPLC-based activity profiling daphnane-type diterpenoid orthoesters (DDOs) 1, 4, and 5 were identified as the active compounds [55.4 ± 7.0% (n = 4), 42.5 ± 16.0% (n = 3), and 51.3 ± 9.4% (n = 4), respectively, at 100 µM]. In a detailed phytochemical profiling of the active extract, 16 compounds were isolated and characterized, including two 2-phenylpyranones (15 and 16) with an unprecedented tetrahydro-4H-5,8-epoxypyrano[2,3-d]oxepin-4-one skeleton, two new DDOs (3 and 4), two new guaiane sesquiterpenoids (11 and 12), and 10 known compounds (1, 2, 5-10, 13, and 14). Structure elucidation was achieved by extensive spectroscopic analysis (1D and 2D NMR, HRMS, and electronic circular dichroism), computational methods, and X-ray crystallography.

HPLC-Based Activity Profiling for hERG Channel Inhibitors in the South African Medicinal Plant Galenia africana.

The human ether-a-go-go-related gene channel is a voltage-activated K(+) channel involved in cardiac action potential. Its inhibition can lead to QT prolongation, and eventually to potentially fatal arrhythmia. Therefore, it is considered a primary antitarget in safety pharmacology. To assess the risk of human ether-a-go-go-related gene channel inhibition by medicinal plants, 700 extracts from different parts of 142 medicinal plants collected in Southern Africa were screened on Xenopus laevis oocytes. A CH2Cl2 extract from the stems and leaves of Galenia africana (Aizoaceae) reduced the peak tail human ether-a-go-go-related gene current by 50.4 ± 5.5 % (n = 3) at a concentration of 100 µg/mL. By means of high-performance liquid chromatography-based activity profiling, nine flavonoids were identified in the active time windows. However, the human ether-a-go-go-related gene channel inhibition of isolated compounds was less pronounced than that of extract and active microfractions (human ether-a-go-go-related gene inhibition between 10.1 ± 5 and 14.1 ± 1.6 at 100 µM). The two major constituents, 7,8-methylenedioxyflavone (1) and 7,8-dimethoxyflavone (13), were quantified (4.3 % and 9.4 %, respectively, in the extract). Further human ether-a-go-go-related gene inhibition tests for compounds 1 and 13 at 300 µM showed a concentration-dependent inhibitory activity (33.2 ± 12.4 and 30.0 ± 7.4, respectively). In a detailed phytochemical profiling of the active extract, a total of 20 phenolic compounds, including six new natural products, were isolated and identified.

Medicinal plants used by traditional healers for the treatment of malaria in the Chipinge district in Zimbabwe.

ETHNOPHARMACOLOGICAL RELEVANCE: Because about 50% of the Zimbabwean population is at risk of contracting malaria each year, the majority of people, especially in rural areas, use traditional plant-based medicines to combat malaria. This explorative ethnobotanical survey was undertaken to document how malaria is conceptualized and diagnosed by traditional healers, and to record the medicinal plants used in the prevention and treatment of malaria, their mode of preparation and administration. MATERIALS AND METHODS: The research was conducted in three villages in Headman Muzite׳s area and in Chiriga village. These villages are located in the Chipinge district in the Manicaland Province in Zimbabwe.Traditional healers were selected with the assistance of the headman of the Muzite area and a representative of the Zimbabwe National Traditional Healers Association. Semi-structured interviews were conducted with 14 traditional healers from four villages in the Chipinge district in Zimbabwe. RESULTS: In total, 28 plants from 16 plant families are used by the healers who manage malaria with medicinal plants. The most cited plant is Cassia abbreviata Oliv. (Leguminosae) followed by Aristolochia albida Duch (Aristolociaceae) and Toddalia asiatica (L.) Lam. (Rutaceae). Roots (55.3%) are the most common part used. Most of the plant parts used to treat malaria are stored as dried powders in closed bottles. The powders are soaked in hot or cold water and the water extract is taken as the active medicine. The healers consider their medicinal knowledge as a spiritual family heritage. Only 25% of the healers refer the malaria patients that do not respond to their treatment to hospital - they believe evil spirits cause their remedies to failure and they would rather try a different plant or perform a cleansing ceremony. CONCLUSIONS: Local knowledge of medicinal plants in the treatment of malaria still exists in all four villages surveyed and traditional healers appear to play an important role in primary health care services in this remote rural area in Zimbabwe. This explorative survey underscores the need to preserve and document traditional healing for managing malaria and for more future scientific research on the plants to determine their efficacy and their safety. This could improve their traditional anti-malarial recipes and might contribute to a better integration of Zimbabwean traditional medicine into the national health system in the future.

Iriflophenone-3-C-glucoside from Cyclopia genistoides: isolation and quantitative comparison of antioxidant capacity with mangiferin and isomangiferin using on-line HPLC antioxidant assays.

The benzophenone, iriflophenone-3-C-glucoside, was isolated from Cyclopia genistoides using a combination of fluid-fluid extraction, high performance counter-current chromatography (HPCCC) and semi-preparative high performance liquid chromatography (HPLC). The microplate oxygen radical absorbance capacity (ORAC) assay, with fluorescein as probe, was adapted for use in an on-line HPLC configuration. The method was validated using a mixture of authentic standards including iriflophenone-3-C-glucoside, and the xanthones, mangiferin and isomangiferin. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was included in the mixture for calculation of Trolox equivalent antioxidant capacity (TEAC) values. Using the on-line HPLC-ORAC assay, as well as 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS(+)) on-line assays, the antioxidant activity of iriflophenone-3-C-glucoside and isomangiferin was demonstrated for the first time. Iriflophenone-3-C-glucoside presented no radical scavenging ability against DPPH, but scavenged ABTS(+) and peroxyl radicals (TEACABTS of 1.04 and TEACORAC of 3.61). Isomangiferin showed slightly lower antioxidant capacity than mangiferin against DPPH (TEACDPPH of 0.57 vs. 0.62), but higher capacity against ABTS(+) (TEACABTS of 1.82 vs. 1.67) and peroxyl radical (TEACORAC of 4.14 vs. 3.69) than mangiferin. The on-line HPLC-ORAC assay was shown to be more sensitive for radical scavengers, but at the same time less selective for rapid radical scavengers than the DPPH assay.

The development and validation of an LC-MS/MS method for the determination of a new anti-malarial compound (TK900D) in human whole blood and its application to pharmacokinetic studies in mice.

BACKGROUND: Malaria is one of the most lethal and life-threatening killer infectious diseases in the world, and account for the deaths of more than half a million people annually. Despite the remarkable achievement made in preventing and eradicating malaria, it still remains a threat to the public health and a burden to the global economy due to the emergence of multiple-drug resistant malaria parasites. Therefore, the need to develop new anti-malarial drugs is crucial. The chemistry department at the University of Cape Town synthesized a number of new CQ-like derivatives (TK-series), and evaluated them for in vitro activity against both CQ-sensitive and -resistant Plasmodium falciparum strains, and for general cytotoxicity against a Chinese Hamster Ovarian (CHO) mammalian cell line. The lead compounds from the TK-series were selected for a comprehensive pharmacokinetic (PK) evaluation in a mouse model. METHODS: A sensitive LC-MS/MS assay was developed for the quantitative determination of TK900D. Multiple reaction monitoring (MRM) in the positive ionization mode was used for detection. The analyte and the internal standard (TK900E) were isolated from blood samples by liquid-liquid extraction with ethyl acetate. Chromatographic separation was achieved with a Phenomenex® Kinetex C18 (100 × 2.0 mm id, 2.6 µm) analytical column, using a mixture of 0.1% formic acid and acetonitrile (50:50; v/v) as the mobile phase. The method was fully validated over concentrations that ranged from 3.910 to 1000 ng/ml, and used to evaluate the PK properties of the lead compounds in a mouse model. RESULTS: The assay was robust, with deviation not exceeding 11% for the intra- and inter-run precision and accuracy. Extraction recovery was consistent and more than 60%. PK evaluation showed that TK900D and TK900E have moderate oral bioavailability of 30.8% and 25.9%, respectively. The apparent half-life ranged between 4 to 6 h for TK900D and 3.6 to 4 h for TK900E. CONCLUSION: The assay was sensitive and able to measure accurately low drug levels from a small sample volume (20 µl). PK evaluation showed that the oral bioavailability was moderate. Therefore, from a PK perspective, the compounds look promising and can be taken further in the drug development process.

Concise and scalable synthesis of aspalathin, a powerful plasma sugar-lowering natural product.

Aspalathin (1), a dihydrochalcone C-glucoside, exhibits powerful plasma sugar-lowering properties and thus potentially could be used to treat diabetes. Small quantities occur in rooibos tea, manufactured via fermentation of the leaves of Aspalathus linearis, hence necessitating the need for an efficient and concise synthesis. Efforts to synthesize aspalathin (1) via coupling of a glucose donor to the nucleophilic phloroglucinol ring of the dihydrochalcone moiety have invariably failed, presumably because of ring deactivation by the electron-withdrawing carbonyl group. Reduction of the carbonyl group of a chalcone (15) and coupling of the resulting 1,3-diarylpropane (16) to tetra-O-benzyl-ß-D-glucopyranose afforded the C-glucosyl-1,3-diarylpropane (17). Regiospecific benzylic oxidation regenerated the carbonyl group and afforded the per-O-methylaspalathin (1a) quantitatively. This method was not successful with the per-O-benzyl-protected dihydrochalcone. However, the nucleophilicity of the phenolic hydroxy groups of the dihydrochalcone or its acetophenone precursor is not diminished by the carbonyl group. Thus, glucosylation of the di-O-benzylacetophenone (5c) at -40 °C afforded the α-O-glucoside (19) in 86% yield. Raising the temperature allowed facile BF3-catalyzed rearrangement to the ß-C-glucoside (6b), which upon hydrogenation, afforded aspalathin (1) in 80% overall yield [based on the usage of di-O-benzylphloroacetophenone (5c) and tetra-O-benzyl-1α-fluoro-ß-D-glucose (2e)].

Analysis of commercial proanthocyanidins. Part 4: solid state (13)C NMR as a tool for in situ analysis of proanthocyanidin tannins, in heartwood and bark of quebracho and acacia, and related species.

(13)C NMR is an effective method of characterizing proanthocyanidin (PAC) tannins in quebracho (Schinopsis lorentzii) heartwood and black wattle (Acacia mearnsii) bark, before and after commercial extraction. The B-rings of the constituent flavan-3-ols, catechols (quebracho) or pyrogallols (wattle), are recognized in unprocessed source materials by "marker" signals at ca. 118 or 105ppm, respectively. NMR allows the minimum extraction efficiency to be calculated; ca. 30%, and ca. 80%, for quebracho heartwood and black wattle bark, respectively. NMR can also identify PAC tannin (predominantly robinetinidin), and compare tannin content, in bark from other acacia species; tannin content decreases in the order A. mearnsii, Acacia pycnantha (87% of A. mearnsii), Acacia dealbata and Acacia decurrens (each 74%) and Acacia karroo (30%). Heartwood from an underexploited PAC tannin source, Searsia lancea, taxonomically close to quebracho, shows abundant profisetinidin and catechin PACs. NMR offers the advantage of being applicable to source materials in their native state, and has potential applications in optimizing extraction processes, identification of tannin sources, and characterization of tannin content in cultivar yield improvement programmes.

Occurrence and sensory perception of Z-2-(ß-d-glucopyranosyloxy)-3-phenylpropenoic acid in rooibos (Aspalathus linearis).

Z-2-(ß-d-glucopyranosyloxy)-3-phenylpropenoic acid (PPAG), a compound postulated to contribute to the taste and mouthfeel of fermented rooibos tea (Aspalathus linearis), was isolated from unfermented rooibos plant material. Its structure was unequivocally confirmed by LC-MS, -MS(2), FT-IR and NMR of the underivatised natural product, and optical rotation measurements of the hydrolysed sugar moiety. A similar compound, postulated to be E-2-(ß-d-glucopyranosyloxy)-3-phenylpropenoic acid, was also detected. Analysis of the leaves of a large number of rooibos plants (n=54), sampled at commercial plantations, showed that PPAG is not ubiquitously present in detectable quantities in the leaves of different plants. This leads to large variation in the fermented plant material, infusions and food-grade extracts. PPAG was shown to have a slightly bitter to astringent taste and a detection threshold of 0.4 mg/l in water.

Analysis of commercial proanthocyanidins. Part 3: the chemical composition of wattle (Acacia mearnsii) bark extract.

Wattle (Acacia mearnsii) bark extract is an important renewable industrial source of natural polymers for leather tanning and adhesive manufacturing. The wattle bark proanthocyanidin oligomers have 5-deoxy extender units that render the interflavanyl bonds resistant to acid catalysed hydrolysis and their composition cannot be determined via conventional thiolysis. We combined established phyto- and synthetic chemistry perspectives with an electrospray mass spectrometry investigation to establish that the flavan-3-ol based oligomers consist of a starter unit which is either catechin or gallocatechin, angularly bonded to fisetinidol or predominantly robinetinidol extender units.

Analysis of commercial proanthocyanidins. Part 2: An electrospray mass spectrometry investigation into the chemical composition of sulfited quebracho (Schinopsis lorentzii and Schinopsis balansae) heartwood extract.

Proanthocyanidins (PACs) are natural plant-derived polymers used in leather tanning, wood adhesives, water purification, and mud additives for oil drilling. Quebracho (Schinopsis lorentzii and Schinopsis balansae) heartwood and mimosa (Acacia mearnsii) bark extracts are the major industrial sources of PACs. These commercial extracts are often sulfited via treatment with sodium hydrogen sulfite to reduce their viscosity and increase their solubility in water. An ESI-MS investigation into the molecular composition of sulfited (cold-water-soluble) quebracho heartwood extract indicates that sulfitation of the PACs occurs via S(N)2 attack of a sulfite ion at both C-2 and C-4 of the constituent flavan-3-ol monomer extender units. Attack at C-2 leads to the opening of the pyran ring. This releases an additional electron-donating phenolic hydroxy group on the A-ring and renders the extract more nucleophilic and suitable for the manufacturing of adhesives. Attack at C-4 leads to interflavanyl bond fission and decrease of the PAC oligomer chain length. The introduction of sulfonic acid moieties at C-2 or C-4 increases the polarity and water solubility of the hot water soluble (unsulfited) extract and transforms it into a cold-water-soluble extract.

Analysis of commercial proanthocyanidins. Part 1: the chemical composition of quebracho (Schinopsis lorentzii and Schinopsis balansae) heartwood extract.

Quebracho (Schinopsis lorentzii and Schinopsis balansae) extract is an important source of natural polymers for leather tanning and adhesive manufacturing. We combined established phyto- and synthetic chemistry perspectives with electrospray mass spectrometry experiments to prove that quebracho proanthocyanidin polymers consist of an homologous series of flavan-3-ol based oligomers. The starter unit is always catechin which is angularly bonded to fisetinidol extender units. By comparison of the MS(2) fragmentation spectra of the oligomer with product ion scans of authentic catechin and robinetinidol samples, we proved that quebracho extract contains no robinetinidol, as is often reported. Quebracho proanthocyanidins have acid resistant interflavanyl bonds, due to the absence of 5-OH groups in fisetinidol, and the aDP cannot be determined via conventional thiolysis and phloroglucinolysis. We used the MS data to estimate a conservative (minimum value) aDP of 3.1.

Tannin fingerprinting in vegetable tanned leather by solid state NMR spectroscopy and comparison with leathers tanned by other processes.

Solid state ¹³C-NMR spectra of pure tannin powders from four different sources--mimosa, quebracho, chestnut and tara--are readily distinguishable from each other, both in pure commercial powder form, and in leather which they have been used to tan. Groups of signals indicative of the source, and type (condensed vs. hydrolyzable) of tannin used in the manufacture are well resolved in the spectra of the finished leathers. These fingerprints are compared with those arising from leathers tanned with other common tanning agents. Paramagnetic chromium (III) tanning causes widespread but selective disappearance of signals from the spectrum of leather collagen, including resonances from acidic aspartyl and glutamyl residues, likely bound to Cr (III) structures. Aluminium (III) and glutaraldehyde tanning both cause considerable leather collagen signal sharpening suggesting some increase in molecular structural ordering. The ²7Al-NMR signal from the former material is consistent with an octahedral coordination by oxygen ligands. Solid state NMR thus provides easily recognisable reagent specific spectral fingerprints of the products of vegetable and some other common tanning processes. Because spectra are related to molecular properties, NMR is potentially a powerful tool in leather process enhancement and quality or provenance assurance.

Photochemistry of flavonoids.

Flavonoids and their photochemical transformations play an important role in biological processes in nature. Synthetic photochemistry allows access to molecules that cannot be obtained via more conventional methods. This review covers all published synthetic photochemical transformations of the different classes of flavonoids. It is first comprehensive review on the photochemistry of flavonoids.

Synthesis of proanthocyanidins. Part 1. The first oxidative formation of the interflavanyl bond in procyanidins.

A novel and efficient method for the oxidative condensation of tetra-O-methyl-3-oxocatechin 4 with tetra-O-methylcatechin is described. Treatment of a solution of 3 (2 equiv) and 4 (1 equiv) with silver tetrafluoroborate readily affords the phenolic per-O-methyl ethers of 3-oxocatechin(4-8)-catechin 18 and 19. Subsequent metal hydride reduction provides access to procyanidin B-3 analogues with the 3,4-cis diastereomers predominating.

Employing atmospheric pressure photoionization in liquid chromatography/tandem mass spectrometry to minimize ion suppression and matrix effects for the quantification of venlafaxine and O-desmethylvenlafaxine.

During the development of a method for quantitative determination of venlafaxine and its major metabolite O-desmethylvenlafaxine, elevated concentrations of the analyte as well as co-eluting matrix compounds caused ion suppression. This ion suppression was inconsistent and therefore influenced the reproducibility of detection. The use of atmospheric pressure photoionization (APPI) in the positive mode was investigated as a tool to circumvent this problem. Employing APPI resulted in negligible ion suppression and increased linearity of the concentration range. A selective, sensitive and rapid liquid chromatography/tandem mass spectrometry method for the determination of venlafaxine and its major metabolite O-desmethylvenlafaxine in human plasma was developed. The analyte was extracted from plasma into tert-butyl methyl ether followed by back extraction into 2% formic acid. An Agilent 1100 high-performance liquid chromatography (HPLC) system, employing reversed-phase chromatography on a cyano column, coupled to an Applied Biosystems API 3000 triple quadrupole mass spectrometer set to multiple reaction monitoring (MRM) mode, was used for separation and detection of the analytes. The method was validated between 2.36-605 ng per mL with a mean recovery of approximately 88% for both parent compound and metabolite analytes. APPI technology was employed to improve the reproducibility of detection enabling rapid, selective and sensitive quantification of venlafaxine and O-desmethylvenlafaxine in human plasma samples.