Bioautography-guided HPTLC-MS as a rapid hyphenated technique for the identification of antimicrobial compounds from selected South African Combretaceae species.

INTRODUCTION: Many species within Combretaceae are traditionally used for the treatment of bacterial infections. The similarity in chemistry and antimicrobial activities within the family pose a challenge in selecting suitable species for herbal drug development. OBJECTIVE: This study aimed at rapidly identifying antimicrobial compounds using bioautography-guided high-performance thin-layer chromatography coupled with mass spectrometry (HPTLC-MS). METHODS: Hierarchical cluster analysis of ultra-performance liquid chromatography-mass spectrometry data from the methanol extracts of 77 samples, representing four genera within Combretaceae, was carried out. Based on groupings on the dendrogram, 15 samples were selected for bioautography analysis against four pathogens (Staphylococcus aureus, Bacillus cereus, Escherichia coli and Salmonella typhimurium). Active compounds were identified using HPTLC-MS analysis of bands corresponding to the inhibition zones. RESULTS: Bioautography revealed 15 inhibition zones against the four pathogens, with the most prominent present for Combretum imberbe. Analysis of the active bands, using HPTLC-MS indicated that flavonoids, triterpenoids and combretastatin B5 contributed to the antibacterial activity. The compounds corresponding to molecular ions m/z 471 (Combretum imberbe) and 499 (Combretum elaeagnoides) inhibited all four pathogens, and were identified as imberbic acid and jessic acid, respectively. Chemotaxonomic analysis indicated that arjunic acid, ursolic acid and an unidentified triterpenoid (m/z 471) were ubiquitous in the Combretaceae species and could be responsible for their antibacterial activities. CONCLUSION: Application of HPTLC-MS enabled the rapid screening of extracts to identify active compounds within taxonomically related species. This approach allows for greater efficiency in the natural product research workflow to identify bioactive compounds in crude extracts.

Antibacterial Screening, Biochemometric and Bioautographic Evaluation of the Non-Volatile Bioactive Components of Three Indigenous South African Salvia Species.

Salvia africana-lutea L., S. lanceolata L., and S. chamelaeagnea L. are used in South Africa as traditional medicines to treat infections. This paper describes an in-depth investigation into their antibacterial activities to identify bioactive compounds. Methanol extracts from 81 samples were screened against seven bacterial pathogens, using the microdilution assay. Biochemometric models were constructed using data derived from minimum inhibitory concentration (MIC) and ultra-performance liquid chromatography-mass spectrometry data. Active molecules in selected extracts were tentatively identified using high-performance thin layer chromatography (HPTLC), combined with bioautography, and finally, by analysis of active zone eluates by mass spectrometry (MS) via a dedicated interface. Salvia chamelaeagnea displayed notable activity towards all seven pathogens, and the activity, reflected by MICs, was superior to that of the other two species, as confirmed through ANOVA. Biochemometric models highlighted potentially bioactive compounds, including rosmanol methyl ether, epiisorosmanol methyl ether and carnosic acid. Bioautography assays revealed inhibition zones against A. baumannii, an increasingly multidrug-resistant pathogen. Mass spectral data of the eluted zones correlated to those revealed through biochemometric analysis. The study demonstrates the application of a biochemometric approach, bioautography, and direct MS analysis as useful tools for the rapid identification of bioactive constituents in plant extracts.

Emodin - A natural anthraquinone derivative with diverse pharmacological activities.

Emodin (1,3,8-trihydroxy-6-methyl-anthraquinone) is a natural anthraquinone derivative that is present in numerous globally renowned herbal medicines. It is recognised as a protein tyrosine kinase inhibitor and as an anticancer drug, active against various tumour cells, including lung, breast, liver, and ovarian cancer cells. Recently, its role in combination chemotherapy with various allopathic medicines, to minimize their toxicity and to enhance their efficacy, has been studied. The use of emodin in these therapies is gaining popularity, due to fewer associated side effects compared with standard anticancer drugs. Emodin has a broad therapeutic window, and in addition to its antineoplastic activity, it displays anti-ulcer, anti-inflammatory, hepatoprotective, neuroprotective, antimicrobial, muscle relaxant, immunosuppressive and antifibrotic activities, in both in vitro and in vivo models. Although reviews on the anticancer activity of emodin have been published, none coherently unite all the pharmacological properties of emodin, particularly the anti-oxidant, antimicrobial, antidiabetic, immunosuppressive and hepatoprotective activities of the compound. Hence, in this review, all of the available data regarding the pharmacological properties of emodin are explored, with particular emphasis on the modes of action of the molecule. In addition, the manuscript details the occurrence, biosynthesis and chemical synthesis of the compound, as well as its toxic effects on biotic systems.

Phytochemical Profiling and Quality Control of Terminalia sericea Burch. ex DC. Using HPTLC Metabolomics.

Terminalia sericea is used throughout Africa for the treatment of a variety of conditions and has been identified as a potential commercial plant. The study was aimed at establishing a high-performance thin layer chromatography (HPTLC) chemical fingerprint for T. sericea root bark as a reference for quality control and exploring chemical variation within the species using HPTLC metabo3lomics. Forty-two root bark samples were collected from ten populations in South Africa and extracted with dichloromethane: methanol (1:1). An HPTLC method was optimized to resolve the major compounds from other sample components. Dichloromethane: ethyl acetate: methanol: formic acid (90:10:30:1) was used as the developing solvent and the plates were visualized using 10% sulfuric acid in methanol as derivatizing agent. The concentrations of three major bioactive compounds, sericic acid, sericoside and resveratrol-3-O-ß-rutinoside, in the extracts were determined using a validated ultra-performance liquid chromatography-photodiode array (UPLC-PDA) detection method. The rTLC software (written in the R-programming language) was used to select the most informative retardation factor (Rf) ranges from the images of the analysed sample extracts. Further chemometric models, including principal component analysis (PCA) and hierarchical cluster analysis (HCA), were constructed using the web-based high throughput metabolomic software. The rTLC chemometric models were compared with the models previously obtained from ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS). A characteristic fingerprint containing clear bands for the three bioactive compounds was established. All three bioactive compounds were present in all the samples, although their corresponding band intensities varied. The intensities correlated with the UPLC-PDA results, in that samples containing a high concentration of a particular compound, displayed a more intense band. Chemometric analysis using HCA revealed two chemotypes, and the subsequent construction of a loadings plot indicated that sericic acid and sericoside were responsible for the chemotypic variation; with sericoside concentrated in Chemotype 1, while sericic acid was more abundant in Chemotype 2. A characteristic chemical fingerprint with clearly distinguishable features was established for T. sericea root bark that can be used for species authentication, and to select samples with high concentrations of a particular marker compound(s). Different chemotypes, potentially differing in their therapeutic potency towards a particular target, could be distinguished. The models revealed the three analytes as biomarkers, corresponding to results reported for UPLC-MS profiling and thereby indicating that HPTLC is a suitable technique for the quality control of T. sericea root bark.

Investigating antimicrobial compounds in South African Combretaceae species using a biochemometric approach.

ETHNOPHARMACOLOGICAL RELEVANCE: Many species within the family Combretaceae are popular medicinal plants that are used traditionally to treat various conditions, of which many are related to bacterial infections. Global concerns regarding the increasing resistance of pathogens towards currently available antibiotics have encouraged researchers to find new drugs with antibacterial activity, particularly from plant sources. AIM OF THE STUDY: This study was aimed at exploring the broad-spectrum antibacterial potential of methanol extracts of species representing four genera of Combretaceae (Combretum, Pteleopsis, Quisqualis, Terminalia), indigenous to South Africa, using a biochemometric approach. MATERIALS AND METHODS: The microdilution assay was used to determine the antibacterial activities, measured as minimum inhibitory concentrations (MICs), of the 51 methanol extracts representing 35 Combretaceae species, against nine species of pathogenic bacteria. Integrative biochemometric analysis was performed, thereby correlating the MIC values with the metabolomic data obtained from ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) analysis. Orthogonal projections to latent structures-discriminant analysis (OPLS-DA) models were constructed for six pathogens displaying variation in their susceptibility towards the extracts. RESULTS: Evaluation of the overall MIC values obtained indicated that extracts of species from the four genera displayed the highest activity towards Bacillus cereus ATCC 11778 (average MIC 0.52 mg/mL) and Salmonella typhimurium ATCC 14028 (average MIC 0.63 mg/mL). These bacteria were the most sensitive Gram-positive and Gram-negative bacteria, respectively. Extracts from Combretum acutifolium, Combretum imberbe and Combretum elaeagnoides were the most active, with average MIC values of 0.70 mg/mL, 0.52 mg/mL and 0.45 mg/mL, respectively. Five triterpenoid compounds were tentatively identified as biomarkers from the biochemometric analysis. CONCLUSION: Correlation of the phytochemistry of species from four genera in the Combretaceae family with antibacterial activity revealed that triterpenoids are responsible for the broad-spectrum antibacterial activity observed.

Chemotypic variation of non-volatile constituents of Artemisia afra (African wormwood) from South Africa.

Artemisia afra (African wormwood) is a popular medicinal plant of southern Africa and is an excellent candidate for commercialisation. This current study was aimed at exploring the phytochemistry and chemical variation of non-volatile compounds within wild populations of A. afra, and developing chromatographic quality control protocols for raw materials based on the identification of marker compounds. Chromatographic data, from samples representing 12 distinct populations, were obtained using liquid chromatography-mass spectrometry. An untargeted chemometric approach revealed three clusters. Marker compounds for each cluster, revealed through discriminant analysis, were isolated and identified using NMR spectroscopy, as acacetin (1) (Group 1), chrysoeriol (2) (Group 2), and 3,5-di-O-caffeoylquinic acid (3) and scopoletin (4) (Group 3). In addition, (3) and rutin (5), (both reported for the first time from A. afra), and (1), (2), (4) and 4-caffeoylquinic acid (6) were established as reliable markers for species identification, since they were abundant in most samples. Quantitative analysis using a validated method established (4) as the dominant compound in the samples (1080-19,600 µg/g dry weight (d.w.)), followed by (5) (49.5-2490 µg/g d.w.). A high performance thin layer chromatography (HPTLC) method was developed. The Rf values and colours of the bands corresponding to the marker compounds were recorded so that these compounds could be easily identified for quality control purposes. Multivariate analysis of the data using the rTLC online application confirmed the presence of different chemical groupings within the samples. It was deduced that quantitative, rather than qualitative differences, characterised the samples. Future research should focus on comparing the efficacy of the various chemical clusters in multi-target biological assays aligned to the traditional use of the plant.

Occurrence, distribution, spatio-temporal variability and source identification of n-alkanes and polycyclic aromatic hydrocarbons in water and sediment from Loskop dam, South Africa.

In this study, the spatial and temporal variations in the levels of C8-C40 n-alkanes and 18 polycyclic aromatic hydrocarbons (PAHs) in water and sediment from Loskop Dam (Mpumalanga Province South Africa), were investigated between 2015 and 2017. In addition, their sources, which have not been well defined, were also studied over the period. This water body is sourced from a historically contaminated water body, the Olifants River, which flows through areas where a range of industrial and agricultural activities take place. Mass crocodile and fish mortalities have been recorded in this aquatic system, and contamination by organic pollutants were highlighted as a contributing factor. The total average n-alkane concentrations in water and sediments ranged from 0.574±00811 to 18.8±1.39 µg/L and 4760±243 to 30700±906 µg/kg, respectively. Similarly, PAHs were detected at total average concentrations of between 0.150±00494 and 49.8±6.86 µg/L in water and 61.6±5.95 to 2618±300 µg/kg. n-Alkane and PAH diagnostic ratios indicated a mixture of sources of these compounds, attributed to terrestrial, submerged and floating plant material, as well as petrogenic and pyrogenic combustion. Inlet, middle and upper segment site clustering was observed with non-metric multidimensional scaling (NMDS) and hierarchical cluster analysis (HCA), mainly driven by the prevalence of PAHs at the inlet sites and n-alkanes in the upper reaches. By using indicator compounds, the sources of contamination could be predicted. The strategy described here can be applied to any water body for continuous long-term monitoring of pollutant levels and to identify sources attributing to water pollution.

Unravelling the Antibacterial Activity of Terminalia sericea Root Bark through a Metabolomic Approach.

Terminalia sericea Burch. ex. DC. (Combretaceae) is a popular remedy for the treatment of infectious diseases. It is widely prescribed by traditional healers and sold at informal markets and may be a good candidate for commercialisation. For this to be realised, a thorough phytochemical and bioactivity profile is required to identify constituents that may be associated with the antibacterial activity and hence the quality of raw materials and consumer products. The aim of this study was to explore the phytochemistry and identify the antibacterial constituents of T. sericea root bark, using a metabolomic approach. The chemical profiles and antibacterial activities of 42 root bark samples collected from three districts in the Limpopo Province, South Africa, were evaluated. Dichloromethane:methanol (1:1) extracts were analysed using ultraperformance liquid chromatography (UPLC)-mass spectrometry (MS), and chemometric models were constructed from the aligned data. The extracts were tested against Bacillus cereus (ATCC 11778), Staphylococcus epidermidis (ATCC 12223), Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 8739), Klebsiella pneumoniae (ATCC 13883), Pseudomonas aeruginosa (ATCC 27853), Shigella sonnei (ATCC 9292) and Salmonella typhimurium (ATCC 14028), using the minimum inhibition microdilution assay. Nine compounds; sericic acid, sericoside, resveratrol-3-O-ß-rutinoside, ellagic acid, flavogallonic acid dilactone, methyl-flavogallonate, quercetin-3-(2''-galloylrhamnoside), resveratrol-3-(6''-galloyl)-O-ß-d-glucopyranoside and arjunetin, were isolated from the root bark. All the compounds, with the exception of sericic acid, sericoside and resveratrol-3-O-ß-rutinoside, were isolated for the first time from the root bark of T. sericea. Chemometric analysis revealed clustering that was not population specific, and the presence of three groupings within the samples, characterised by sericic acid, sericoside and an unidentified compound (m/z 682/4.66 min), respectively. The crude extracts from different populations displayed varied antibacterial activities against S. typhimurium (minimum inhibitory concentrations (MICs) 0.25-1.0 mg/mL), but similar activity towards Bacillus cereus (1.0 mg/mL). Several compounds present in the root bark were highly active towards all or most of the pathogens tested, but this activity was not reflected by the chemical profiles of extracts prepared from the individual samples. Among the pure compounds tested, only flavogallonic acid dilactone and methyl-flavogallonate exhibited broad-spectrum activity. A biochemometric analysis indicated that there was no consistent association between the levels of phytochemicals and the activity of the active or non-active extracts. Although it was deduced that the major constituents of T. sericea root bark contributed to the chemotypic variation, further investigation of the interactions of compounds present in the root bark may provide antibacterial efficacies not evident when examining compounds singularly. The data reported herein will provide information that is fundamentally important for the development of quality control protocols.

Isolation, in vitro evaluation and molecular docking of acetylcholinesterase inhibitors from South African Amaryllidaceae.

Inhibition of acetylcholinesterase (AChE) is considered a promising strategy for the treatment of Alzheimer's disease (AD) and dementia. Members of the Amaryllidaceae family are well known for their pharmacologically active alkaloids, including galanthamine, which is used to treat AD. The aim of this study was to evaluate the potential of South African Amaryllidaceae species to inhibit AChE, to isolate the active compounds, and probe their ability to bind the enzyme using molecular docking. The AChE inhibitory activity of extracts of 41 samples, representing 14 genera and 28 species, as well as isolated compounds, were evaluated in vitro using a qualitative thin layer chromatography (TLC) bio-autography assay and Ellman's method in a quantitative 96-well microplate assay. Targeted isolation of compounds was achieved with the aid of preparative-high perfomance liquid chromatography-mass spectrometry. The structures of the isolates were elucidated using nuclear magnetic resonance spectrocopy, and were docked into the active site of AChE to rationalise their biological activities. The most active species were found to be Amaryllis belladonna L (IC50 14.3 ± 2.6 µg/mL), Nerine huttoniae Schönland (IC50 45.3 ± 0.4 µg/mL) and Nerine undulata (L.) Herb. (IC50 52.8 ± 0.5 µg/mL), while TLC bio-autography indicated the presence of several active compounds in the methanol extracts. Four compounds, isolated from A. belladonna, were identified as belladine, undulatine, buphanidrine and acetylcaranine. Acetylcaranine and undulatine were previously isolated from A. belladonna, while belladine and buphanidrine were reported from other South African Amaryllidaceae species. Using Ellman's method, acetylcaranine was found to be the most active of the isolates towards AChE, with an IC50 of 11.7 ± 0.7 µM, comparable to that of galanthamine (IC50 = 6.19 ± 2.60 µM). Molecular docking successfully predicted the binding modes of ligands within receptor binding sites. Acetylcaranine was predicted by the docking workflow to have the highest activity, which corresponds to the in vitro results. Both qualitative and quantitative assays indicate that several South African Amaryllidaceae species are notable AChE inhibitors.

Speciation of major and trace elements leached from coal fly ash and the kinetics involved.

The combustion of coal in thermal power plants may result in high concentrations of elements in the coal fly ash remaining that may be toxic to living organisms or pose a risk to the environment. This study was aimed at determining the concentrations of potentially toxic elements in coal fly ash leachates, in an attempt to simulate natural processes that influence the environment. Powder X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and elemental composition studies were employed to characterize the physicochemical properties of the parent coal fly ash. The effect of various leaching parameters, including the pH of leaching solutions, the volume ratio of leaching solutions to the mass of coal fly ash, leaching time and temperature, were investigated. Moreover, the kinetics of the leaching of toxic elements from coal fly ash was also investigated by considering that the leaching process is governed by dissolution. Zero order, pseudo-first order, pseudo-second order, power function, simple Elovich and parabolic diffusion kinetic models were used to evaluate the leaching process. The experimental results indicate that the pH and leaching time had the most significant effect on the leaching behavior of elements from coal fly ash.

Effect of pH on the toxicity of fumonisins towards the RTL-W1 cell line and zebrafish (Danio rerio) embryos.

Fumonisins are common contaminants of maize. The neutral red assay, using the RTL-W1 trout liver cell line, and the fish embryo test (FET), using zebrafish, were selected to assess the effect of pH on the cytotoxicity, acute toxicity and teratogenicity of fumonisin B1 (FB1). The results demonstrated that FB1 exerts low cytotoxicity towards RTL-W1 cells without pH adjustment (IC50 1 746 µM), and no cytotoxicity after pH-adjustment to physiological conditions. The LC50 value for FB1 in the FET (1 058 µM at 48 h) confirmed low acute toxicity. Adjusting the pH to physiological conditions reduced the acute toxicity of FB1 towards zebrafish embryos, emphasising the importance of acidity/basicity of the medium in toxicity testing. Hydrolysed FB1 was less toxic than FB1 (LC50 2 690 µM at 48 h), and neither were teratogenic towards zebrafish embryos. Results confirm that the FET may account for effects not observable in cell cultures.

Exploring Common Culinary Herbs and Spices as Potential Anti-Quorum Sensing Agents.

Quorum sensing controls bacterial pathogenesis and virulence; hence, interrupting this system renders pathogenic bacteria non-virulent, and presents a novel treatment for various bacterial infections. In the search for novel anti-quorum sensing (AQS) compounds, 14 common culinary herbs and spices were screened for potential antipathogenicity activity against Chromobacterium violaceum ATCC 12472. Extracts of Glycyrrhiza glabra (liquorice), Apium graveolens (celery), Capsicum annuum (cayenne pepper) and Syzygium anisatum (aniseed) demonstrated good AQS potential, yielding opaque halo zones ranging from 12⁻19 mm diameter at sub-minimum inhibitory concentrations (0.350⁻4.00 mg/mL). For the same species, the percentage reduction in violacein production ranged from 56.4 to 97.3%. Zones with violacein inhibitory effects were evident in a celery extract analysed using high performance thin layer chromatography-bio-autography. The major active compound was isolated from celery using preparative-high performance liquid chromatography-mass spectrometry and identified using gas chromatography-mass spectrometry (GC-MS) as 3-n-butyl-4,5-dihydrophthalide (sedanenolide). Potent opaque zones of inhibition observed on the HPTLC-bio-autography plate seeded with C. violaceum confirmed that sedanenolide was probably largely responsible for the AQS activity of celery. The bacteriocidal properties of many herbs and spices are reported. This study, however, was focussed on AQS activity, and may serve as initial scientific validation for the anti-infective properties ascribed to several culinary herbs and spices.

GC-MS profiling of fatty acids in green coffee (Coffea arabica L.) beans and chemometric modeling for tracing geographical origins from Ethiopia.

BACKGROUND: This study was aimed at the development of objective analytical method capable of verifying the production region of the coffee beans. One hundred samples of green coffee (Coffea arabica L.) beans from the major producing regions, comprising various sub-regional types, were studied for variations in their fatty acid compositions by using gas chromatography coupled with mass spectrometry. Principal component analysis (PCA) was used to visualize data trends. Linear discriminant analysis (LDA) was used to construct classification models. RESULTS: Twenty-one different fatty acids were detected in all of the samples. The total fatty acid content varied from 83 to 204 g kg-1 across the regions. Oleic, linoleic, palmitic, stearic and arachidic acids were identified as the most discriminating compounds among the production regions. The recognition and prediction abilities of the LDA model for classification at regional level were 95% and 92%, respectively, and 92% and 85%, respectively, at sub-regional level. CONCLUSION: Fatty acids contain adequate information for use as descriptors of the cultivation region of coffee beans. Chemometric methods based on fatty acid composition can be used to detect fraudulently labeled coffees, with regard to the production region. These can benefit the coffee production market by providing consumers with products of the expected quality. © 2019 Society of Chemical Industry.

1H-NMR and UPLC-MS metabolomics: Functional tools for exploring chemotypic variation in Sceletium tortuosum from two provinces in South Africa.

Sceletium tortuosum (Aizoaceae) is widely recognised for the treatment of stress, anxiety and depression, as well as for obsessive compulsive disorders. A comprehensive intraspecies chemotypic variation study, using samples harvested from two distinct regions of South Africa, was done using both proton nuclear magnetic resonance (1H-NMR) spectroscopy of methanol extracts (N = 145) and ultra performance liquid chromatography-mass spectrometry (UPLC-MS) of acid/base extracts (N = 289). Chemometric analysis of the 1H-NMR data indicated two main clusters that were region-specific (Northern Cape and Western Cape provinces). Two dimensional (2D) NMR was used to identify analytes that contributed to the clustering as revealed by the S-plot. The sceletium alkaloids, pinitol and two alkylamines, herein reported for the first time from S. tortuosum, were identified as markers that distinguished the two groups. Relative quantification of the marker analytes conducted by qNMR indicated that samples from the Northern Cape generally contained higher concentrations of all the markers than samples from the Western Cape. Quantitative analysis of the four mesembrine alkaloids using a validated UPLC-photo diode array (PDA) detection method confirmed the results of qNMR with regard to the total alkaloid concentrations. Samples from the Northern Cape Province were found to contain, on average, very high total alkaloids, ranging from 4938.0 to 9376.8 mg/kg dry w. Regarding the Western Cape samples, the total yields of the four mesembrine alkaloids were substantially lower (averages 16.4-4143.2 mg/kg). Hierarchical cluster analysis of the UPLC-MS data, based on the alkaloid chemistry, revealed three branches, with one branch comprising samples primarily from the Northern Cape that seemed somewhat chemically conserved, while the other two branches represented mainly samples from the Western Cape. The construction of an orthogonal projections to latent structures-discriminant analysis model and subsequent loadings plot, allowed alkaloid markers to be identified for each cluster. The diverse sceletium alkaloid chemistry of samples from the three clusters may facilitate the recognition of alkaloid profiles, rather than individual compounds, that exert targeted effects on various brain receptors involved in stress, anxiety or depression.

Identification, Isolation and Determination of Biomarkers for Quality Control of Bush Tea (Athrixia phyllicoides).

Athrixia phylicoides, known as "bush tea", grows abundantly in South Africa. An infusion of the leaves is used as a beverage and to treat a multitude of health conditions. The aim of this study was to investigate the chemical variation within A. phylicoides and to identify characteristic compounds for quality control. Samples from 12 locations in South Africa were analysed using ultra-performance liquid chromatography. Hierarchical cluster analysis of the aligned ultra-performance liquid chromatography-time-of-flight mass spectrometry data indicated two groups on the resulting dendrogram, representing 48 samples. Five marker compounds, identified through visual inspection and the construction of a discriminant analysis model, were evident on the ultra-performance liquid chromatography-MS profiles. Four of these compounds were isolated and identified, three as hydroxy methoxyflavones and the fourth as a coumarate, using nuclear magnetic resonance spectroscopy. An ultra-performance liquid chromatography-photodiode array method was developed and validated for the determination of the marker compounds using the isolates as standards. The limits of detection for the four compounds ranged from 0.92 - 2.50 µg/mL. Their recoveries at three concentration levels (1.00, 10.0, and 100 µg/mL) were between 97.0 and 101%, while acceptable intra- and inter-day precision was obtained as reflected by percentage relative standard deviation values below 2.24%. The concentrations of all the marker compounds were found to be higher in samples corresponding to Group 1 of the dendrogram than in those from Group 2. This may be attributable to differences in altitude, climate, and some edaphic factors. Identification of these marker compounds will make a valuable contribution towards the quality control and sustainable commercialisation of bush tea.

Potential Herb-Drug Pharmacokinetic Interactions between African Wild Olive Leaf Extract and Selected Antihypertensive Drugs.

The African wild olive (Olea europaea subsp. africana) is traditionally used as a hypotensive agent. Herb-drug interactions may result from the concurrent use of herbal medicines and conventional prescription drugs. This aspect was investigated by determining the effect of the extract on the in vitro intestinal epithelial permeation of selected hypotensive drugs using the Caco-2 cell culture model. The phytochemical profiles of leaf extracts of African wild olive from different localities in South Africa were compared, since efficacy is determined by the chemical composition. Extracts were analysed using ultra-performance liquid chromatography. The oleuropein concentration varied considerably from below the detection limit (4.94 µg/mL) to 59.4 mg/g dry weight. Chemometric models constructed from the aligned chromatographic data indicated only quantitative differences between the profiles. The leaf extract was found to increase the permeability of propranolol in the absorptive direction (Papp = 8.93 × 10-6 cm/s) across Caco-2 cell monolayers, but considerably decreased transport in the secretory direction (Papp = 3.68 × 10-6 cm/s). The permeation of diltiazem was enhanced by the extract in both the absorptive (Papp = 7.33 × 10-6 cm/s) as well as in the secretory direction (Papp = 7.16 × 10-6 cm/s), but a decrease in the efflux ratio was observed. The extract therefore caused a net increase in the transport of both drugs in the absorptive direction due to an inhibition effect on their efflux. This suggests a potential increase in the blood levels of these drugs when taken simultaneously with African wild olive leaf extract, indicating potential adverse effects that must be verified in vivo.

Single-step isolation of embelin using high-performance countercurrent chromatography and determination of the fatty acid composition of seeds of Embelia schimperi.

Embelin (2,5-dihydroxy-3-undecyl-p-benzoquinone) is known for its potent anthelmintic activity, but also for wound-healing, antidiabetic, anticonvulsant, antitumour, anti-inflammatory, analgesic, hepatoprotective, antioxidant, antibacterial and antispermatogenic activities. A high-performance countercurrent chromatography method was developed for the purification of embelin from an extract of the seeds of Embelia schimperi fruit. The optimized solvent system (n-hexane-ethylacetate-ethanol-water, 7:3:7:3) resulted in the isolation of 13.9 mg of embelin, directly from 100 mg of crude extract, in a single step within a short time (40 min). Although the compound appeared to be completely pure when analysed by ultra-performance liquid chromatography (UPLC) with photo diode array detection, the purity was established as ~90% by UPLC-mass spectrometry. Furthermore, we report the fatty acid composition of the seeds of E. schimperi fruit. Nine fatty acids were quantified from the fruit seed extract by gas chromatography-mass spectrometry, with linoleic (46.4%), palmitic (21.5%) and oleic (19.6%) acids making up the largest proportions.

Isolation and in vitro permeation of phenylpropylamino alkaloids from Khat (Catha edulis) across oral and intestinal mucosal tissues.

ETHNOPHARMACOLOGICAL RELEVANCE: Khat, the leaves of Catha edulis, is used as a "natural amphetamine-like" stimulant in eastern and southern Africa, as well as in the Arabian Peninsula. Leaves are masticated to elicit a state of euphoria. Although the psychostimulatory effects of the leaves are attributed to the presence of phenylpropylamino alkaloids (i.e. cathinone, cathine and norephedrine), the extent of permeation of these alkaloids across the oral and intestinal mucosa has not been established. MATERIALS AND METHODS: Cathinone was isolated in the form of the oxalate salt from young buds, following acid-base extraction. High performance countercurrent chromatography (HPCCC) was used to isolate cathine and norephedrine, following borohydride reduction of a mixture of the three alkaloids. The in vitro permeability of these three alkaloids in their pure form, as well as in a crude extract, was evaluated across Caco-2 cell monolayers and across excised porcine intestinal, sublingual and buccal tissues. RESULTS: The purities of the isolated cathine and norephedrine were in excess of 90%, thereby proving that HPCCC can be applied for efficient separation of these alkaloids from extracts of Khat. The apparent permeability (Papp) coefficients for the Khat alkaloids in their pure form were all above 1.0×10-6cm/s, indicating that the transport of the three alkaloids across the selected biological membranes is comparable to that of the highly permeable reference compound, caffeine. Although readily transported across the various membranes, the alkaloids were transported to a lesser extent when present in a leaf extract, suggesting that other phytochemicals present in the extract influence their permeation. CONCLUSIONS: These results provide evidence that chewing of Khat contributes to the buccal and sublingual absorption of the psychoactive alkaloids in the bloodstream directly across the oral mucosal membranes. In addition, it confirms that these metabolites will be readily absorbed from the gastrointestinal tract when swallowed.

Rapid analysis of the skin irritant p-phenylenediamine (PPD) in henna products using atmospheric solids analysis probe mass spectrometry.

Henna (Lawsonia inermis) is applied to stain keratin, present in hair, skin and fingernails, a red-orange or rust colour. Producers of temporary tattoos mix the aromatic amine compound, para-phenylenediamine (PPD) into natural henna to create 'black henna' that rapidly stains the skin black. However, PPD may cause severe delayed hypersensitivity reactions following skin contact. This study proposes a rapid direct-analysis method to detect and identify PPD using an atmospheric solids analysis probe (ASAP) coupled to a Q-ToF mass spectrometer (MS). Since laborious, multistep methods of analysis to determine PPD are undesirable, due to the instability of the compound in solution, a screening method involving no sample preparation steps was developed. Experiments were carried out to optimise the corona current, sample cone voltage, source temperature, and desolvation gas temperature to determine ideal ASAP-Q-ToF-MS analysing conditions. Eleven of the 109 henna samples, originating from various countries, tested positive for PPD when henna products were screened using ASAP-MS, without any form of sample preparation other than grinding. Ultra-performance liquid chromatography electrospray ionisation-mass spectrometry (UPLC-Q-ToF-MS) was subsequently used to confirm the results from ASAP and to determine the concentrations of PPD in henna products. The allergen was detected in the same eleven samples, with concentrations ranging from 0.05-4.21% (w/w). It can be concluded that the sensitivity of the ASAP-MS technique is sufficient (limit of detection=0.025% w/w) to allow screening of henna samples for the presence of PPD. This relatively new technique can be applied to commercial products without extraction, sample treatment or chromatographic separation.

Myricetin: A Dietary Molecule with Diverse Biological Activities.

Myricetin is a common plant-derived flavonoid and is well recognised for its nutraceuticals value. It is one of the key ingredients of various foods and beverages. The compound exhibits a wide range of activities that include strong anti-oxidant, anticancer, antidiabetic and anti-inflammatory activities. It displays several activities that are related to the central nervous system and numerous studies have suggested that the compound may be beneficial to protect against diseases such as Parkinson's and Alzheimer's. The use of myricetin as a preserving agent to extend the shelf life of foods containing oils and fats is attributed to the compound's ability to protect lipids against oxidation. A detailed search of existing literature revealed that there is currently no comprehensive review available on this important molecule. Hence, the present work includes the history, synthesis, pharmaceutical applications and toxicity studies of myricetin. This report also highlights structure-activity relationships and mechanisms of action for various biological activities.