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Antimicrobial research into the use of Melaleuca alternifolia essential oil has demonstrated broad-spectrum activity; however, much of the research published focuses on identifying the potential of this essential oil individually, rather than in combination for an enhanced antimicrobial effect. This study aimed to determine the antimicrobial activity of four essential oil combinations, all inclusive of M. alternifolia, against nine pathogens associated with the respiratory tract. The minimum inhibitory concentration assay was used to determine the antimicrobial activity of four essential oil combinations, M. alternifolia in combination with Cupressus sempervirens, Origanum majorana, Myrtus communis, and Origanum vulgare essential oils. The interactions between essential oil combinations were analyzed using isobolograms and SynergyFinder 2.0 software to visualize the synergistic potential at varied ratios. The antimicrobial activity of the different combinations of essential oils all demonstrated the ability to produce an enhanced antimicrobial effect compared to the essential oils when investigated independently. The findings of this study determined that isobolograms provide a more in-depth analysis of an essential oil combination interaction; however, the value of that interaction should be further quantified using computational modelling such as SynergyFinder. This study further supports the need for more studies where varied ratios of essential oils are investigated for antimicrobial potential.

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.

Hypoxis hemerocallidea (African potato) is a popular medicinal plant that has been used traditionally for the treatment of various disorders. Some HIV/AIDS patients use this traditional medicine together with their antiretroviral therapy. This study aimed to determine the impact of selected H. hemerocallidea materials (i.e., a commercial product, an aqueous extract, and biomass reference plant material) on the bidirectional permeability of indinavir across Caco-2 cell monolayers as well as the bioavailability of indinavir during an acute, single administration study in Sprague-Dawley rats. All of the selected H. hemerocallidea test materials demonstrated inhibition effects on indinavir efflux across Caco-2 cell monolayers, albeit to different extents. An increase in the bioavailability of indinavir was obtained in vivo when administered concomitantly with the H. hemerocallidea materials, albeit not statistically significantly. The change in bioavailability directly correlated with the in vitro permeability results. It can therefore be concluded that the change in permeability and bioavailability of indinavir was caused by efflux inhibition and this effect was dependent on the type of H. hemerocallidea material investigated, which was found to be in the following order: commercial product > aqueous extract > reference plant material. The clinical significance of the combined effect of efflux and metabolism inhibition by H. hemerocallidea should be determined in another in vivo model that expresses the cytochrome P450 3A4 enzyme.

Foot odour (bromodosis) is an embarrassing and perplexing condition mostly caused by bacteria of the Brevibacterium species. Essential oils are a credible option as an affordable treatment of odour and contribute towards antimicrobial efficacy. Therefore, this study sets out to investigate the antimicrobial activity of essential oil combinations against odour-causing bacteria. The broth microdilution method was used to investigate the antimicrobial activity of 119 essential oil combinations, and the fractional inhibitory index was calculated to determine the interactive profile. Combinations that resulted in synergy in 1 : 1 ratios were further evaluated in different concentrations, and isobolograms were plotted to determine the influence of the ratio on overall activity. Numerous combinations could be identified as having synergistic interactions against the Brevibacterium spp. and no antagonism was observed. The combination of Juniperus virginiana (juniper) and Styrax benzoin (benzoin) demonstrated synergy against all three Brevibacterium spp. tested and J. virginiana was the essential oil responsible for the majority of the synergistic interactions. The results reported here confirm the promising potential of the majority of these oils and selected combinations in treating and controlling bromodosis.

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.

Actaea racemosa (black cohosh) has a history of traditional use in the treatment of general gynecological problems. However, the plant is known to be vulnerable to adulteration with other cohosh species. This study evaluated the use of shortwave infrared hyperspectral imaging (SWIR-HSI) in tandem with chemometric data analysis as a fast alternative method for the discrimination of four cohosh species (Actaea racemosa, Actaea podocarpa, Actaea pachypoda, Actaea cimicifuga) and 36 commercial products labelled as black cohosh. The raw material and commercial products were analyzed using SWIR-HSI and ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) followed by chemometric modeling. From SWIR-HSI data (920 – 2514 nm), the range containing the discriminating information of the four species was identified as 1204 – 1480 nm using Matlab software. After reduction of the data set range, partial least squares discriminant analysis (PLS-DA) and support vector machine discriminant analysis (SVM-DA) models with coefficients of determination (R2 ) of ≥ 0.8 were created. The novel SVM-DA model showed better predictions and was used to predict the commercial product content. Seven out of 36 commercial products were recognized by the SVM-DA model as being true black cohosh while 29 products indicated adulteration. Analysis of the UHPLC-MS data demonstrated that six commercial products could be authentic black cohosh. This was confirmed using the fragmentation patterns of three black cohosh markers (cimiracemoside C; 12-β,21-dihydroxycimigenol-3-O-L-arabinoside; and 24-O-acetylhydroshengmanol-3-O-β-D-xylopyranoside). SWIR-HSI in conjunction with chemometric tools (SVM-DA) could identify 80% adulteration of commercial products labelled as black cohosh.

Scutellaria lateriflora (skullcap) is a medicinal herb that has a long history of use in the treatment of ailments such as insomnia and anxiety. Commercial herbal formulations claiming to contain S. laterifolia herba have flooded the consumer markets. However, due to intentional or unintentional adulteration, cases of hepatotoxicity have been reported. Possible adulteration with the potentially hepatotoxic Teucrium spp., T. canadense and T. chamaedrys has been reported. In this study, hyperspectral imaging in combination with multivariate image analysis methods was used to differentiate S. laterifolia, T. canadense , and T. chamaedrys raw materials in a non-destructive manner. Furthermore, the ability to detect adulteration of raw materials using the developed multivariate models was also investigated. Chemical images were captured using a shortwave infrared pushbroom imaging system in the wavelength range 920–2514 nm. Principal component analysis was applied to the images to investigate chemical differences between the species. Partial least squares discriminant analysis was used to model pre-assigned class images, and the classification model predicted the levels of adulteration in spiked raw materials. UHPLC-MS as an independent analytical technique was used to confirm chemical differences between the three species. The ability of hyperspectral chemical imaging as a non-destructive technique in the differentiation of the three species was achieved with three distinct clusters in the score scatter plot. A 92.3 % variation in modelled data using PC1 and PC2 was correlated to chemical differences between the three species. Near infrared signals in the regions 1924 nm and 2092 nm (positive P1), 1993 nm and 2186 nm (negative P1), 1918 nm, 2092 nm, and 2266 nm (positive P2), as well as 1993 nm and 2303 nm (negative P2) were identified as containing discriminating information using the loadings line plots. Chemical imaging of spiked samples showed spatial orientation of contaminants within the powdered samples, and percentage adulteration was accurately predicted at levels ≥ 40 % adulteration based on pixel abundance.

The buccal mucosal route offers several advantages but the delivery of certain drugs can be limited by low membrane permeability. This study investigated the buccal permeability properties of didanosine (ddI) and assessed the potential of Aloe vera gel (AVgel) as a novel buccal permeation enhancer. Permeation studies were performed using Franz diffusion cells, and the drug was quantified by UV spectroscopy. Histomorphological evaluations were undertaken using light and transmission electron microscopy. The permeability of ddI was concentration-dependent, and it did not have any adverse effects on the buccal mucosae. A linear relationship (R² = 0.9557) between the concentrations and flux indicated passive diffusion as the mechanism of drug transport. AVgel at concentrations of 0.25 to 2 %w/v enhanced ddI permeability with enhancement ratios from 5.09 (0.25 %w/v) to 11.78 (2 %w/v) but decreased permeability at 4 and 6 %w/v. Ultrastructural analysis of the buccal mucosae treated with phosphate buffer saline pH 7.4 (PBS), ddI/PBS, and ddI/PBS/AVgel 0.5 %w/v showed cells with normal plasmalemma, well-developed cristae, and nuclei with regular nuclear envelopes. However, cells from 1, 2, and 6 %w/v AVgel-treated mucosae showed irregular nuclear outlines, increased intercellular spacing, and plasmalemma crenulations. This study demonstrates the potential of AVgel as a buccal permeation enhancer for ddI to improve anti-HIV and AIDS therapy.

Hoodia gordonii is a spiny succulent plant popularly consumed for its purported anti-obesity effect. Traditionally used by the Khoi-San of South Africa and Namibia as a hunger and thirst suppressant while on long hunting trips, the commercialisation of this plant has been highly controversial due to intellectual property rights and benefit sharing issues, as well as the fact that several prominent pharmaceutical companies involved in its development have withdrawn their interest. Quality control has been the main focus of scientific studies as the supply of H. gordonii plant material is limited due to its sparse geographical distribution, slow maturation rate, need for a permit to cultivate or export material as well as high public demand, contributing to adulteration of a large amount of products. Despite the isolation of numerous steroidal glycosides from H. gordonii, the main focus has been on the pregnane glycoside P57, considered to be the active ingredient and marker molecule to determine quality of raw material and products. Publications based on scientific studies of key aspects such as in vivo biopharmaceutics, the biological activity of all chemical constituents, clinical efficacy, and especially safety are insufficient or completely absent causing great concern as H. gordonii is one of the most widely consumed anti-obesity products of natural origin. This review offers an up-to-date overview of all the current available knowledge pertaining to H. gordonii achieved by systematic analysis of the available literature.

The effect of Aloe vera (L.) Burm. f. (Aloe barbadensis Miller) gel and whole leaf extract on the permeability of Caco-2 cell monolayers was determined. Solutions of gel and the whole leaf extract were applied to the cell monolayers, and the transepithelial electrical resistance was monitored for 2 hours, which was then continued for another 2 hours after removal of the test solutions to measure reversibility of the effect. The transport of insulin in the presence and absence of the A. vera gel and whole leaf extract solutions was also investigated. Both the A. vera gel and whole leaf extract were able to significantly reduce the transepithelial electrical resistance of the Caco-2 cell monolayers at concentrations above 0.5 % w/v and thereby showed the ability to open tight junctions between adjacent cells. This effect was fully reversible, as the electrical resistance of the cell monolayers returned to the original value upon removal of the test solutions. The A. vera gel and whole leaf extract solutions significantly enhanced the transport of insulin across the Caco-2 cell monolayers compared with the control. The results suggest that these plant products have a high potential to be used as absorption enhancers in novel dosage forms for drugs with poor bioavailabilities when administered orally. On the other hand, an uncontrolled increase in the bioavailability of drugs that are taken simultaneously with A. vera gel and whole leaf extract products may result in adverse effects, and the potential exists that toxic blood plasma levels may be reached.

Harpagophytum procumbens and H. zeyheri (Pedaliaceae) are widely distributed in southern Africa. They are highly valued traditional medicines used in the treatment of inflammation and pain-related ailments such as rheumatoid arthritis, ulcers and boils [1]. The biological activity of the species has been ascribed to the presence of iridoid glycosides such as harpagoside, harpagide and procumbide. However, only harpagoside is currently used as a 'biomarker' to determine the quality of raw material and commercial products. Harpagophytum procumbens is regarded as more commercially valuable as it contains higher levels of the biologically active constituent, harpagoside, however H. zeyheri is being used as an adulterant to meet high market demands [2]. To explore the extent of variation, three hundred samples were collected throughout the geographical distribution of the two species and the harpagoside content quantified using LC-MS. The results confirm that both species contain harpagoside and that it is present in higher amounts in H. procumbens with a range of 0.17 – 4.37%, whereas in H. zeyheri, the range was 0.00 – 3.07%. According to the standard set by the European Pharmacopoeia, the harpagoside content is required to be at least 1.2%. All of the H. procumbens samples tested contained harpagoside, although only 61 met the 1.2% specification. Only 38 H. zeyheri samples contained harpagoside and only 26 met the specifications indicating that it is not a suitable substitute. This study confirmed that the two taxa are highly variable and intricate species as reflected by the significant variation in harpagoside content. This study provides valuable information regarding the difficulties of quality control and can also be used as a basis for the selection of raw material for propagation. Acknowledgements: The National Research Foundation of South Africa is thanked for its financial contribution to the study. References: [1] Van Wyk B-E, Gericke NP (2000) Peoples Plants. A guide to Useful Plants of Southern Africa. Briza Publications, Pretoria, South Africa. [2] Stewart KM, Cole D, (2005)J Ethnopharmacol, 100: 225 – 236.

It is a well-known fact that 80% of the world's population use herbal medicines. The herbal medicines market has increased dramatically in recent years, as consumers became more health conscious, herbal medicines are considered safe by consumers and these products are generally less expensive than allopathic medicines. These products are however not rigorously regulated and quality and safety cannot be guaranteed. In fact, many cases of toxicity have been reported, in some cases due to errors in species identification that is, or should be, one of the first steps in an herbal quality control protocol. Established, methodical processes are necessary to standardize herbal medicines to produce consistent and reproducible products. Herbal medicines pose significant standardization and quality control challenges due to their innate phytochemical complexity and intraspecies variability. Standardization and quality control involves inter alia the authentication of plants followed by the quantification of identified biomarkers and the selection and cultivation of the chemotype with the desired characteristics (e.g. ratios of biomarkers). Vibrational spectroscopy has been identified and implemented as an important quality control technique in the pharmaceutical, food and beverage and agricultural industries. The technique has gained popularity as it is non-destructive, inexpensive, and any sample matrix (powder, liquid, essential oils) can be tested with minimal or no sample preparation. Therefore, no bias is introduced as a result of using different extraction solvents and the entire metabolome is used. The large datasets obtained using vibrational spectroscopy (NIR, MIR and hyperspectral imaging) are analyzed using chemometric data analysis. This allows for species authentication using discrimination models constructed using the Orthogonal Projections to Latent Structures (OPLS) technique. In combination with data obtained from standard analytical methods (e.g. LC-MS), calibration models that correlates the information in the spectral data to the chemical or reference data was used to quantify biomarkers. In addition, cluster formation during exploratory principle component analysis supplies valuable information through the detection of similarities between samples. This may be used in conjunction with hierarchical cluster analysis to identify and suggest the best chemotype to cultivate. Several examples related to quality control and standardization of medicinally and commercially important plant species will be discussed: Species authentication and/or biomarker quantification (Agathosma, Harpagophytum, Illicium and Pelargonium species); chemotype selection (Sceletium tortuosum).