Metabolomic profiling of wild rooibos (Aspalathus linearis) ecotypes and their antioxidant-derived phytopharmaceutical potential.

INTRODUCTION: Aspalathus linearis (commonly known as rooibos) is endemic to the Cape Floristic Region of South Africa and is a popular herbal drink and skin phytotherapeutic ingredient, with health benefits derived primarily from its unique phenolic content. Several, seemingly habitat-specific ecotypes from the Cederberg (Western Cape) and Northern Cape have morphological, ecological, genetic and biochemical differences. OBJECTIVES AND METHODS: Despite the commercial popularity of the cultivated variety, the uncultivated ecotypes are largely understudied. To address gaps in knowledge about the biochemical constituency, ultra-performance liquid chromatography-mass spectrometry analysis of fifteen populations was performed, enabling high-throughput metabolomic fingerprinting of 50% (v/v) methanolic extracts. Antioxidant screening of selected populations was performed via three assays and antimicrobial activity on two microbial species was assessed. The metabolomic results were corroborated with total phenolic and flavonoid screening of the extracts. RESULTS AND DISCUSSION: Site-specific chemical lineages of rooibos ecotypes were confirmed via multivariate data analyses. Important features identified via PLS-DA disclosed higher relative abundances of certain tentative metabolites (e.g., rutin, aspalathin and apiin) present in the Dobbelaarskop, Blomfontein, Welbedacht and Eselbank sites, in comparison to other locations. Several unknown novel metabolites (e.g., m/z 155.0369, 231.0513, 443.1197, 695.2883) are responsible for metabolomic separation of the populations, four of which showed higher amounts of key metabolites and were thus selected for bioactivity analysis. The Welbedacht and Eselbank site 2 populations consistently displayed higher antioxidant activities, with 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging activities of 679.894 ± 3.427 µmol Trolox/g dry matter and 635.066 ± 5.140 µmol Trolox/g dry matter, respectively, in correlation with a high number of phenolic and flavonoid compounds. The contribution of the individual metabolites to the pharmacological effectiveness of rooibos remains unknown and as such, further structural elucidation and phytopharmacological testing is thus urgently needed.

Stevens' Cure (Umckaloabo)-the vindication of a patent medicine.

Stevens' Cure (Umckaloabo) emerged as a patent medicine claiming to treat tuberculosis in the United Kingdom at the beginning of the 20th century. However, due to its identity being shrouded in secrecy, it was never truly accepted by the medical community. It was "rediscovered" in the 1970s and subsequently developed into a very popular and successful phytopharmaceutical for the treatment of upper respiratory tract infections. Whether Stevens' Cure contained the same ingredient(s) as the modern Umckaloabo has not yet been demonstrated. We attempted to elucidate for the first time the identity of the original ingredient by comparative analysis of historical product samples. Three historical samples of Stevens' Cure were compared with Pelargonium sidoides DC. and P. reniforme Curt. root per UPLC-MS analysis. We confirm that the ingredient-P. sidoides DC.-is indeed the same as used in modern phytotherapy. We also attribute the first ethnopharmacological record of P. sidoides DC. being used for the treatment of tuberculosis to C. H. Stevens, the "creator" of Umckaloabo.

Fatal pyrrolizidine alkaloid poisoning of infants caused by adulterated Senecio coronatus.

Senecio coronatus (known as izonkozonko and ubulibazi in Zulu) is commonly used in traditional medicine in South Africa as purification purgative and enemas for infants during weaning. We show for the first time that this species does not contain pyrrolizidine alkaloids and that reported cases of fatal hepatic sinusoidal obstruction syndrome in infants were caused by wrongly identified Senecio species containing large amounts of retrorsine-N-oxide. A validated ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the detection and quantitation of pyrrolizidine alkaloids is described.

Assessment of glutathione levels in model solution and grape ferments supplemented with glutathione-enriched inactive dry yeast preparations using a novel UPLC-MS/MS method.

A novel, robust and fast ultra-high performance liquid chromatography-MS method has been developed for the simultaneous quantification of reduced glutathione (GSH) and oxidised glutathione (GSSG) in grape juice, wine and model wine solution. Sample preparation is minimal and does not require derivatisation. The method has very good performance in terms of sensitivity and selectivity. The limit of detection was 0.002 and 0.001 mg L(-1) for GSH and GSSG, respectively. The amount of GSH and GSSG released by commercial glutathione-enriched inactivated dry yeast preparations (GSH-IDYs) into a model solution was assessed. Significant differences in the amount of GSH and/or GSSG released into a model wine by different GSH-IDYs were observed, with ethanol influencing this release under certain conditions. The GSH and GSSG levels in grape juice fermentations supplemented with GSH-IDY were also assessed in relation to different addition times during fermentation. GSH-IDY addition can lead to elevated wine GSH levels, provided the supplementation is done early during alcoholic fermentation.

Resistance in Maize Inbred Lines to Fusarium verticillioides and Fumonisin Accumulation in South Africa.

Fusarium ear rot of maize, caused by Fusarium verticillioides, is an important disease affecting maize production worldwide. Apart from reducing yield and grain quality, F. verticillioides produces fumonisins which have been associated with mycotoxicoses of animals and humans. Currently, no maize breeding lines are known with resistance to F. verticillioides in South Africa. The objective of this study, therefore, was to evaluate 24 genetically diverse maize inbred lines as potential sources of resistance to Fusarium ear rot and fumonisin accumulation in field trials at Potchefstroom and Vaalharts in South Africa. After artificial silk channel inoculation with F. verticillioides, Fusarium ear rot development was determined at harvest and fumonisins B1, B2, and B3 quantified. A significant inbred line by location effect was observed for Fusarium ear rot severity (P ≤ 0.001), although certain lines proved to be consistently resistant across both locations. The individual inbred lines also differed considerably in fumonisin accumulation between Potchefstroom and Vaalharts, with differentiation between susceptible and potentially resistant inbred lines only being possible at Vaalharts. A greenhouse inoculation trial was then also performed on a subset of potentially resistant and highly susceptible lines. The inbred lines CML 390, CML 444, CML 182, VO 617Y-2, and RO 549 W consistently showed a low Fusarium ear rot (<5%) incidence at both Potchefstroom and Vaalharts and in the greenhouse. Two of these inbred lines, CML 390 and CML 444, accumulated fumonisin levels <5 mg kg-1. These lines could potentially act as sources of resistance for use within a maize breeding program.

Quantification of melamine absorption, distribution to tissues, and excretion by sheep.

Eight Döhne Merino rams were used to quantify apparent absorption, distribution to tissues, and excretion of dietary melamine in sheep. Two batches of concentrate pellets were made; one (CON) contained corn gluten meal with no detectable melamine and the other (MEL) contained corn gluten meal that was previously found to be highly contaminated with melamine at 15,117 mg/kg. The MEL pellets contained 1,149 mg/kg of melamine. During a 10-d adaptation period, all the animals received a forage-based diet supplemented with 600 g/d of the CON pellets. This was followed by an 8-d collection period during which 6 of the animals received MEL pellets and 2 received CON pellets. Melamine intake of sheep that received MEL pellets was 0.69 g/d. Blood samples were taken before first ingestion of MEL pellets on d 1 and again on d 3, 6, and 8 of the collection period for melamine and serum creatinine analyses. Feces and urine were collected quantitatively over the 8 d for proximate and melamine analyses. All the animals were slaughtered at the end of the trial, and samples of the LM, liver, kidneys, and abdominal fat were taken for melamine analysis. Data of the 2 sheep that received CON pellets for the duration of the trial confirmed that no melamine was detected in any of the samples, and no statistical analyses were performed on these data. The apparent digestibility or efficiency of absorption of ingested melamine was 76.7%. Melamine was detected in the urine, blood, muscle (LM), and fat tissue of all the sheep that received MEL pellets. Serum melamine concentrations reached 5.4 mg/kg on d 8 of the collection period, and the meat (LM) contained 9.6 mg/kg of melamine. Calculations on the partitioning of ingested melamine suggested that urine is the major excretion route accounting for 53.2%, whereas feces accounted for 23.3% of ingested melamine. Approximately 3.5% of the ingested melamine was detected in muscle. It was concluded that ingested melamine is highly absorbable from the small intestine and that a pathway exists for the distribution of dietary melamine to meat.

Hot topic: pathway confirmed for the transmission of melamine from feed to cow's milk.

Eight lactating Holstein cows were randomly allotted to 2 groups in a trial to establish whether a pathway exists for the transmission of melamine from feed to milk. All cows received oat hay ad libitum and 15 kg of concentrate pellets per cow daily. The concentrate pellets contained either melamine-contaminated corn gluten meal of Chinese origin (melamine treatment) or locally produced melamine-free corn gluten meal (control treatment). Cows in the melamine treatment ingested 17.1 g of melamine per day. Cows were milked twice daily, and milk samples were taken once daily during the afternoon milking for melamine and milk component analyses. Melamine appeared in the milk within 8 h after first ingestion of the melamine containing pellets. Melamine concentration reached a maximum of 15.7 mg/kg within 56 h after first ingestion, with an excretion efficiency of approximately 2%. Milk solids and milk urea nitrogen were not affected by treatment. The melamine concentration dropped rapidly after changing all cows back to the control pellets, but melamine only declined to undetectable levels in the milk more than 6 d (152 h) after last ingestion of melamine. Results from the current trial are important to the feed and dairy industries because, until now, any melamine found in milk and milk products was attributed only to the deliberate external addition of melamine to these products, not to adulterated ingredients in animal feeds.

Mammalian exocrine secretions. XVII: chemical characterization of preorbital secretion of male suni, Neotragus moschatus.

Gas chromatographic and gas chromatographic-mass spectrometric techniques were employed to identify 83 compounds, including alkanes, alkenes, aldehydes, 2-methylalkanes, carboxylic acids, 1-alkyl formates and alken-1-yl formates, benzoic acid, and cholesterol, in the preorbital secretion of the male suni, Neotragus moschatus. Dimethyl disulfide derivatization and lithium aluminum hydride reduction were used to determine the position of double bonds and to confirm the identity of the functional groups in some of the constituents of the secretion.

Toxicokinetics of ochratoxin A in vervet monkeys (Cercopithecus aethiops).

The toxicokinetics of ochratoxin A were investigated in vervet monkeys (Cercopithecus aethiops). Three female monkeys were treated intravenously with ochratoxin A at doses, respectively, of 0.8, 1.5 and 2 mg/ kg body weight (BW). Blood and urine samples were collected over a period of 21 days. Plasma and urine extracts were analysed by high-performance liquid chromatography (HPLC) with either fluorescence or negative ion electrospray ionization mass spectrometric detection. The clearance of ochratoxin A from plasma followed a two-compartment model. The elimination half-life of ochratoxin A in the monkeys was determined to be 19-21 days and the average total body clearance was 0.22 +/- 0.07 ml/h per kg and the average apparent distribution volume of the central compartment was 59 +/- 9 ml/kg and the peripheral compartment was 59 +/- 20 ml/kg. No evidence was found for any metabolic conversion of ochratoxin A.

A kinetic study into the hydrolysis of the ochratoxins and analogues by carboxypeptidase A.

The hydrolyses of the ochratoxins and analogues by carboxypeptidase A were assessed. This was done by measuring the amount of phenylalanine formed with liquid chromatography coupled to tandem electrospray mass spectrometry. The kinetic data of ochratoxin A, ochratoxin B, and the synthetic bromo-ochratoxin B were compared to the values of a number of synthesized structure analogues, namely, ochratoxin A methyl ester, ochratoxin B methyl ester, N-(2-hydroxybenzoyl)phenylalanine, N-(5-chloro-2-hydroxybenzoyl)phenylalanine, N-(5-bromo-2-hydroxybenzoyl)phenylalanine, and N-(5-fluoro-2-hydroxybenzoyl)phenylalanine. The halogen-containing analogues had lower turnovers than their des-halo analogues. There are no substantial differences in the kinetic data between the different halogen-containing analogues.

Screening of commercial hydrolases for the degradation of ochratoxin A.

Ochratoxin A (OTA) is a nephrotoxic and carcinogenic mycotoxin. The toxin is a common contaminant of various foods and feeds and poses a serious threat to the health of both humans and animals. A number of commercial hydrolases were screened for the ability to degrade OTA to nontoxic compounds. A crude lipase from Aspergillus niger (Amano A) proved to substantially hydrolyze OTA to the nontoxic OTalpha and phenylalanine, as confirmed by HPLC with fluorescence detection. The enzyme was purified by anion exchange chromatography to homogeneity. Activity staining of the purified enzyme with alpha-naphthyl acetate/Fast Red revealed only one band exhibiting hydrolytic activity. The specific activity of the purified enzyme toward OTA was 2.32 units/mg.

Influence of halogen salts on the production of the ochratoxins by Aspergillus ochraceus Wilh.

The first report of the biological production of bromo ochratoxin B by Aspergillus ochraceus Wilh. is presented as well as a study of the influence of potassium bromide, potassium iodide, potassium fluoride, and potassium chloride on the production of ochratoxin A and ochratoxin B. Potassium fluoride and potassium iodide inhibited the growth of the fungus, whereas potassium chloride substantially stimulated the production of ochratoxin A in shaken solid substrate fermentation on whole wheat or shredded wheat, generally giving a high yield of ochratoxins. Increasing levels of potassium bromide led to a decline in ochratoxin A production and an increase in bromo-ochratoxin B, ochratoxin B, and 4-hydroxy ochratoxin B. Nevertheless, A. ochraceus was much less versatile in the bromo analogues than other fungi, which produce metabolites containing chlorine. Analysis included aminopropyl solid-phase extraction column cleanup followed by quantitative analysis on reversed-phase HPLC using fluorescence detection and employing N-(5-chloro-2-hydroxybenzoyl)phenylalanine as an internal standard.