Characterisation of Sclerocarya birrea (marula) seed oil and investigation of the geographical origin by applying similarity calculations, differential NMR and hierarchical cluster analysis.

INTRODUCTION: The marula fruit is an important indigenous African fruit since various commercial products are produced from the pulp and the seed oil. The increased demand requires methods for authentication, quality control and determination of geographical origin. OBJECTIVE: The study aimed to establish a fast and reliable method for characterisation and authentication of marula seed oil. Furthermore, to identify marker compounds that can distinguish marula seed oils from other commercial oils and indicate regional differences. MATERIALS AND METHODS: Metabolic profiling of 44 commercial marula seed oils was performed using proton nuclear magnetic resonance (1 H NMR). For rapid classification similarity calculations were compared with principal component analysis. Differential NMR was used to determine marker compounds. RESULTS: Marula seed oil was found to be similar to macadamia and olive oils and was distinguished from these oils by the detection of minor components. Marula seed oil is differentiated from the other two oils by the absence of α-linolenic acid, relatively high levels of monoglycerides and diglycerides, and an approximately 1:1 ratio of 1,2- and 1,3-diglycerides. When comparing marula seed oils from various regions using hierarchical cluster analysis, clustering of the marula seed oils from Namibia and Zimbabwe was observed and was related to the quantities of linoleic acid and monoglycerides and diglycerides. Some samples displayed deviations in their composition which might indicate adulteration or contamination during the production process. CONCLUSION: The study demonstrates the potential of NMR as a tool in the quality control of marula seed oil. This technique requires very little sample preparation, circumvents derivatisation of the oil components with fast run-times. In addition, samples with chemical profiles that differ from the general signature profile can easily be identified.

Permeation of Phytochemicals of Selected Psychoactive Medicinal Plants across Excised Sheep Respiratory and Olfactory Epithelial Tissues.

The intranasal route of drug administration offers an opportunity to bypass the blood-brain barrier and deliver compounds directly into the brain. Scientific evidence exists for medicinal plants (e.g., Centella asiatica and Mesembryanthemum tortuosum) to treat central nervous system conditions such as anxiety and depression. The ex vivo permeation of selected phytochemicals (i.e., asiaticoside and mesembrine) has been measured across excised sheep nasal respiratory and olfactory tissue. Permeation studies were conducted on individual phytochemicals and C. asiatica and M. tortuosum crude extracts. Asiaticoside exhibited statistically significantly higher permeation across both tissues when applied alone as compared to the C. asiatica crude extract, while mesembrine permeation was similar when applied alone or as M. tortuosum crude extract. Permeation of all the phytocompounds was similar or slightly higher than that of the drug atenolol across the respiratory tissue. Permeation of all the phytocompounds was similar to or slightly lower than that of atenolol across the olfactory tissue. In general, the permeation was higher across the olfactory epithelial tissue than across the respiratory epithelial tissue and therefore showed potential for direct nose-to-brain delivery of the selected psychoactive phytochemicals.

Optimizing the Antimicrobial Synergism of Melaleuca alternifolia (Tea Tree) Essential Oil Combinations for Application against Respiratory Related Pathogens.

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.

The African cherry: A review of the botany, traditional uses, phytochemistry, and biological activities of Prunus africana (Hook.f.) Kalkman.

ETHNOPHARMACOLOGICAL RELEVANCE: Prunus africana (Hook.f.) Kalkman (Rosaceae), commonly known as "Pygeum" or "African cherry", occurs in mainland montane forests scattered across sub-Saharan Africa, Madagascar, and some surrounding islands. Traditionally, decoctions of the stem-bark are taken orally for the treatment of a wide variety of conditions, such as benign prostatic hyperplasia (BPH), stomach ache, chest pain, malaria, heart conditions, and gonorrhoea, as well as urinary and kidney diseases. The timber is used to make axe handles and for other household needs. The dense wood is also sawn for timber. AIM: The fragmented information available on the ethnobotany, phytochemistry, and biological activities of the medicinally important P. africana was collated, organised, and analysed in this review, to highlight knowledge voids that can be addressed through future research. MATERIALS AND METHODS: A bibliometric analysis of research output on P. africana was conducted on literature retrieved, using the Scopus® database. The trend in the publications over time was assessed and a network analysis of collaborations between countries and authors was carried out. Furthermore, a detailed review of the literature over the period 1971 to 2021, acquired through Scopus, ScienceDirect, SciFinder, Pubmed, Scirp, DOAJ and Google Scholar, was conducted. All relevant abstracts, full-text articles and various book chapters on the botanical and ethnopharmacological aspects of P. africana, written in English and German, were consulted. RESULTS: A total of 455 documents published from 1971 to 2021, were retrieved using the Scopus search. Analysis of the data showed that the majority of these documents were original research articles, followed by reviews and lastly a miscellaneous group comprising conference papers, book chapters, short surveys, editorials and letters. Data were analysed for annual output and areas of intense research focus, and countries with high research output, productive institutions and authors, and collaborative networks were identified. Prunus africana is reported to exhibit anti-inflammatory, analgesic, antimicrobial, anti-oxidant, antiviral, antimutagenic, anti-asthmatic, anti-androgenic, antiproliferative and apoptotic activities amongst others. Phytosterols and other secondary metabolites such as phenols, triterpenes, fatty acids, and linear alcohols have been the focus of phytochemical investigations. The biological activity has largely been ascribed to the phytosterols (mainly 3-ß-sitosterol, 3-ß-sitostenone, and 3-ß-sitosterol-glucoside), which inhibit the production of prostaglandins in the prostate, thereby suppressing the inflammatory symptoms associated with BPH and chronic prostatitis. CONCLUSIONS: Many of the ethnobotanical assertions for the biological activity of P. africana have been confirmed through in vitro and in vivo studies. However, a disparity exists between the biological activity of the whole extract and that of single compounds isolated from the extract, which were reported to be less effective. This finding suggests that a different approach to biological activity studies should be encouraged that takes all secondary metabolites present into consideration. A robust technique, such as multivariate biochemometric data analysis, which allows for a holistic intervention to study the biological activity of a species is suggested. Furthermore, there is a need to develop rapid and efficient quality control methods for both raw materials and products to replace the time-consuming and laborious methods currently in use.

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.

Optimization of Antioxidant Synergy in a Polyherbal Combination by Experimental Design.

Culinary herbs and spices are known to be good sources of natural antioxidants. Although the antioxidant effects of individual culinary herbs and spices are widely reported, little is known about their effects when used in combination. The current study was therefore undertaken to compare the antioxidant effects of crude extracts and essential oils of some common culinary herbs and spices in various combinations. The antioxidant interactions of 1:1 combinations of the most active individual extracts and essential oils were investigated as well as the optimization of various ratios using the design of experiments (DoE) approach. The 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), and ferric reducing antioxidant power (FRAP) assays were used to determine the antioxidant activity, and MODDE 9.1® software (Umetrics AB, Umea, Sweden) was used to determine the DoE. The results revealed synergism for the following combinations: Mentha piperita with Thymus vulgaris methanol extract (ΣFIC = 0.32 and ΣFIC = 0.15 using the DPPH and FRAP assays, respectively); Rosmarinus officinalis with Syzygium aromaticum methanol extract (ΣFIC = 0.47 using the FRAP assay); T. vulgaris with Zingiber officinalis methanol extracts (ΣFIC = 0.19 using the ABTS assay); and R. officinalis with Z. officinalis dichloromethane extract (ΣFIC = 0.22 using the ABTS assay). The DoE produced a statistically significant (R2 = 0.905 and Q2 = 0.710) model that was able to predict extract combinations with high antioxidant activities, as validated experimentally. The antioxidant activities of the crude extracts from a selection of culinary herbs and spices were improved when in combination, hence creating an innovative opportunity for the future development of supplements for optimum health.

Mesembryanthemum tortuosum L. alkaloids modify anxiety-like behaviour in a zebrafish model.

ETHNOPHARMACOLOGICAL RELEVANCE: Mesembryanthemum tortuosum L. (previously known as Sceletium tortuosum (L.) N.E. Br.) is indigenous to South Africa and traditionally used to alleviate anxiety, stress and depression. Mesembrine and its alkaloid analogues such as mesembrenone, mesembrenol and mesembranol have been identified as the key compounds responsible for the reported effects on the central nervous system. AIM OF THE STUDY: To investigate M. tortuosum alkaloids for possible anxiolytic-like effects in the 5-dpf in vivo zebrafish model by assessing thigmotaxis and locomotor activity. MATERIALS AND METHODS: Locomotor activity and reverse-thigmotaxis, recognised anxiety-related behaviours in 5-days post fertilization zebrafish larvae, were analysed under simulated stressful conditions of alternating light-dark challenges. Cheminformatics screening and molecular docking were also performed to rationalize the inhibitory activity of the alkaloids on the serotonin reuptake transporter, the accepted primary mechanism of action of selective serotonin reuptake inhibitors. Mesembrine has been reported to have inhibitory effects on serotonin reuptake, with consequential anti-depressant and anxiolytic effects. RESULTS: All four alkaloids assessed decreased the anxiety-related behaviour of zebrafish larvae exposed to the light-dark challenge. Significant increases in the percentage of time spent in the central arena during the dark phase were also observed when larvae were exposed to the pure alkaloids (mesembrenone, mesembrenol, mesembrine and mesembrenol) compared to the control. However, mesembrenone and mesembranol demonstrated a greater anxiolytic-like effect than the other alkaloids. In addition to favourable pharmacokinetic and physicochemical properties revealed via in silico predictions, high-affinity interactions characterized the binding of the alkaloids with the serotonin transporter. CONCLUSIONS: M. tortuosum alkaloids demonstrated an anxiolytic-like effect in zebrafish larvae providing evidence for its traditional and modern day use as an anxiolytic.

An acute dose-ranging evaluation of the antidepressant properties of Sceletium tortuosum (Zembrin®) versus escitalopram in the Flinders Sensitive Line rat.

ETHNOPHARMACOLOGICAL RELEVANCE: Sceletium tortuosum (L.) N.E.Br. (ST) has been used by the Khoisan people of South Africa as a mood elevator. Its various pharmacological mechanisms of action suggest distinct potential as an antidepressant. Clinical studies in healthy individuals suggest beneficial effects on mood, cognition, and anxiety. AIM OF THE STUDY: To obtain a chromatographic fingerprint of a standardized extract of S. tortuosum (Zembrin®), and to evaluate the acute antidepressant-like properties of Zembrin® versus the reference antidepressant, escitalopram, in the Flinders Sensitive Line (FSL) rat, a genetic rodent model of depression. MATERIALS AND METHODS: The chemical profile of Zembrin® was determined by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) chromatogram method using alkaloid standards. Twelve saline treated FSL and six Flinders Resistant Line (FRL) control rats were used to confirm face validity of the FSL model using the forced swim test (FST). Thereafter, FSL rats (n = 10) received either 5, 10, 25, 50 or 100 mg/kg of Zembrin®, or 5, 10 or 20 mg/kg escitalopram oxalate (ESC), both via oral gavage, and subjected to the open field test (OFT) and FST. RESULTS: Four main ST alkaloids were identified and quantified in Zembrin® viz. mesembrenone, mesembrenol, mesembrine, and mesembranol (47.9%, 32%, 13.2%, and 6.8% of the total alkaloids, respectively). FSL rats showed significantly decreased swimming and climbing (coping) behaviours, and significantly increased immobility (despair), versus FRL controls. ESC 5 mg/kg and Zembrin® 25 mg/kg and 50 mg/kg showed significant dose-dependent reversal of immobility in FSL rats and variable effects on coping behaviours. Zembrin® 50 mg/kg was the most effective antidepressant dose, showing equivalence to ESC 5. CONCLUSIONS: Zembrin® (25 and 50 mg/kg) and ESC (5 mg/kg) are effective antidepressants after acute treatment in the FST, as assessed in FSL rats. Moreover, Zembrin® 50 mg/kg proved equivalent to ESC 5. Further long-term bio-behavioural studies on the antidepressant properties of Zembrin® are warranted.

Exploring four South African Croton species for potential anti-inflammatory properties: in vitro activity and toxicity risk assessment.

ETHNOPHARMACOLOGICAL RELEVANCE: The African Continent harbours approximately 26 Croton species. Many Croton species are used in traditional medicine in southern Africa to treat a variety of ailments including malaria, tuberculosis, microbial infection and inflammation. Considering the high diversity of the genus Croton, the ethnopharmacological information available on southern African species is rather limited. Furthermore, the potential for novel anti-inflammatory drug scaffolds has not previously been investigated. AIM OF THE STUDY: The aim of the study was to evaluate the potential of four South African Croton species extracts (Croton gratissimus, Croton pseudopulchellus, Croton sylvaticus, and Croton steenkampianus) for anti-inflammatory activity targeting the TLR4 signalling pathway and to assess the potential risk for hepatotoxicity and genotoxicity using an in vitro cellomics approach. MATERIAL AND METHODS: Leaf extracts of C. gratissimus, C. pseudopulchellus, C. sylvaticus and C. steenkampianus were prepared using methanol and chloroform (1:1, v/v). The anti-inflammatory activity was determined using LPS induced nitric oxide production in RAW 264.7 macrophages, while the hepatotoxicity and genotoxicity was evaluated using multi-parameter end point analysis in C3A and Vero cells, respectively. Mitochondrial membrane potential, mitochondrial mass, oxidative stress, lysosomal content and lipid accumulation were used as markers to assess the risk for hepatotoxicity. RESULTS: All four species attenuated nitric oxide production with negligible cytotoxicity. However, C. gratissimus yielded the most favorable profile. Cell density was significantly reduced in both C3A and Vero cells with the C. gratissimus extract providing a suitable toxicity profile amenable to further high content analysis. While there was no meaningful effect on mitochondrial dynamics, a strong dose dependent increase in lipid content, paralleled by an expansion of the lysosomal compartment, identifies a potential risk for steatosis. Risk for genotoxicity was investigated using the micronucleus assay which revealed a dose dependent increase in micronuclei formation. Changes in nuclear morphology and cell ploidy further strengthens the associated risk for genotoxicity and suggests the extract from C. gratissimus may function as an aneugen. Collectively, the data demonstrates that although the selected species possess anti-inflammatory components, the risk for possible hepatotoxic and genotoxic side effects may negate their prospect towards further drug development.

Evaluation of the wound healing properties of South African medicinal plants using zebrafish and in vitro bioassays.

ETHNOPHARMACOLOGICAL RELEVANCE: In South Africa, medicinal plants have a history of traditional use, with many species used for treating wounds. The scientific basis of such uses remains largely unexplored. AIM OF THE STUDY: To screen South African plants used ethnomedicinally for wound healing based on their pro-angiogenic and wound healing activity, using transgenic zebrafish larvae and cell culture assays. MATERIALS AND METHODS: South African medicinal plants used for wound healing were chosen according to literature. Dried plant material was extracted using six solvents of varying polarities. Pro-angiogenesis was assessed in vivo by observing morphological changes in sub-intestinal vessels after crude extract treatment of transgenic zebrafish larvae with vasculature-specific expression of a green fluorescent protein. Subsequently, the in vitro anti-inflammatory, fibroblast proliferation and collagen production effects of the plant extracts that were active in the zebrafish angiogenesis assay were investigated using murine macrophage (RAW 264.7) and human fibroblast (MRHF) cell lines. RESULTS: Fourteen plants were extracted using six different solvents to yield 84 extracts and the non-toxic (n=72) were initially screened for pro-angiogenic activity in the zebrafish assay. Of these plant species, extracts of Lobostemon fruticosus, Scabiosa columbaria and Cotyledon orbiculata exhibited good activity in a concentration-dependent manner. All active extracts showed negligible in vitro toxicity using the MTT assay. Lobostemon fruticosus and Scabiosa columbaria extracts showed noteworthy anti-inflammatory activity in RAW 264.7 macrophages. The acetone extract of Lobostemon fruticosus stimulated the most collagen production at 122% above control values using the MRHF cell line, while all four of the selected extracts significantly stimulated cellular proliferation in vitro in the MRHF cell line. CONCLUSIONS: The screening of the selected plant species provided valuable preliminary information validating the use of some of the plants in traditional medicine used for wound healing in South Africa. This study is the first to discover through an evidence-based pharmacology approach the wound healing properties of such plant species using the zebrafish as an in vivo model.

The use of chemometric modelling to determine chemical composition-antimicrobial activity relationships of essential oils used in respiratory tract infections.

The antimicrobial effects of essential oils are commonly cited within aromatherapeutic texts for use in respiratory tract infections. These essential oils are inhaled or applied to the skin to treat infections and manage symptoms associated with these conditions. A limited number of these essential oils have been scientifically studied to support these claims, specifically, against respiratory pathogens. This study reports on the minimum inhibitory concentration (MIC) of 49 commercial essential oils recommended for respiratory tract infections, and identifies putative biomarkers responsible for the determined antimicrobial effect following a biochemometric workflow. Essential oils were investigated against nine pathogens. Three essential oils, Amyris balsamifera (amyris), Coriandrum sativum (coriander) and Santalum austrocaledonicum (sandalwood) were identified as having greater activity (MIC value = 0.03-0.13 mg/ml) compared to the other essential oils investigated. The essential oil composition of all 49 oils were determined using Gas Chromatography coupled to Mass Spectroscopy (GC-MS) analysis and the GC-MS data analysed together with the antimicrobial data using chemometric tools. Eugenol was identified as the main biomarker responsible for antimicrobial activity in the majority of the essential oils. The ability of a chemometric model to accurately predict the active and inactive biomarkers of the investigated essential oils against pathogens of the respiratory tract was 80.33%.

A review of biological activities and phytochemistry of six ethnomedicinally important South African Croton species.

ETHNOPHARMACOLOGICAL RELEVANCE: The genus Croton (Euphorbiaceae) encompasses 1300 species, which consist of a variety of trees and shrubs distributed across the world. About 26 species are harboured on the African continent. This genus plays an essential role in African folk medicine. Croton species are traditionally used for the treatment of many diverse conditions such as diabetes, malaria, sexually transmitted diseases, cancer, inflammation, fever, digestive problems and fungal infections. AIM OF THE REVIEW: To provide a comprehensive overview of the ethnobotany, traditional uses, phytochemistry and biological activities of six selected southern Africa Croton species; C.gratissimus Burch., C. megalobotrys Müll.-Arg., C. menyhartii Gȕrke, C. pseudopulchellus Pax, C. steenkampianus Gerstner and C. sylvaticus Schltdl.). MATERIALS AND METHODS: Various electronic databases, namely Google, Google Scholar, Scopus, ScienceDirect, Biomed Central and Pubmed, were used to search for information related to the traditional uses, chemistry and pharmacology of Croton species. Books were also consulted to collect all pertinent information. RESULTS AND DISCUSSION: The ethnopharmacology, phytochemistry and biological activities of southern African Croton species are reviewed. The literature revealed that Croton species are trusted traditional medicines for the treatment of microbial infections and malaria. The non-volatile components of Croton species include flavonoids, terpenoids and alkaloids, while the volatile constituents comprise mainly of monoterpenes (α-phellandrene, α-pinene and 1,8-cineole) and sesquiterpenes (caryophyllene oxide). Most of the reported biological activities (anti-oxidant, antimicrobial, anticancer and antimalarial) were based on in vitro assays and were accredited to various extracts. However, both in vitro and in vivo studies, linking the reported activities to specific compounds, are still lacking. CONCLUSIONS: Croton species are used in traditional medicine to treat a range of ailments, and various in vitro biological activities have been investigated, with some extracts exhibiting good activity that could be considered for further investigation. The in vitro activities obtained seem to justify the use of Croton species in traditional medicine. Data on in vivo studies are scarce and studies usually focused on a single collection. The need to establish a quality control protocols for the standardisation of these herbal drugs is also important.

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.

Anti-seizure activity of African medicinal plants: The identification of bioactive alkaloids from the stem bark of Rauvolfia caffra using an in vivo zebrafish model.

ETHNOPHARMACOLOGICAL RELEVANCE: Epilepsy is one of the major chronic diseases that does not have a cure to date. Adverse drug reactions have been reported from the use of available anti-epileptic drugs (AEDs) which are also effective in only two-thirds of the patients. Accordingly, the identification of scaffolds with promising anti-seizure activity remains an important first step towards the development of new anti-epileptic therapies, with improved efficacy and reduced adverse effects. Herbal medicines are widely used in developing countries, including in the treatment of epilepsy but with little scientific evidence to validate this use. In the search for new epilepsy treatment options, the zebrafish has emerged as a chemoconvulsant-based model for epilepsy, mainly because of the many advantages that zebrafish larvae offer making them highly suitable for high-throughput drug screening. AIM OF THE STUDY: In this study, 20 medicinal plants traditionally used in South Africa to treat epilepsy were screened for anti-epileptic activity using a zebrafish larvae model. MATERIALS AND METHODS: Toxicity triaging was conducted on 120 crude extracts, 44 fractions and three isolated compounds to determine the maximum tolerated concentration (MTC) of each extract, fraction or compound. MTC values were used to guide the concentration range selection in bioactivity studies. The effectiveness of crude extracts, fractions and isolated compounds from Rauvolfia caffra Sond. in suppression of pentylenetetrazole (PTZ) induced seizure-like behaviour in a 6-dpf zebrafish larvae model was measured using the PTZ assay. RESULTS: Following a preliminary toxicity triage and bioactivity screen of crude extracts from 20 African plants used traditionally for the treatment and management of epilepsy, the methanolic extract of Rauvolfia caffra Sond. was identified as the most promising at suppressing PTZ induced seizure-like behaviour in a zebrafish larvae model. Subsequent bioactivity-guided fractionation and spectroscopic structural elucidation resulted in the isolation and identification of two tryptoline derivatives; a previously unreported alkaloid to which we assigned the trivial name rauverine H (1) and the known alkaloid pleiocarpamine (2). Pleiocarpamine was found to reduce PTZ-induced seizures in a dose-dependent manner. CONCLUSIONS: Accordingly, pleiocarpamine represents a promising scaffold for the development of new anti-seizure therapeutic compounds. Furthermore, the results of this study provide preliminary evidence to support the traditional use of Rauvolfia caffra Sond. in the treatment and management of epilepsy. These findings warrant further studies on the anti-epileptic potential of Rauvolfia caffra Sond. using other models.

Anti-tyrosinase activity of South African Aloe species and isolated compounds plicataloside and aloesin.

Tyrosinase is the key enzyme in the production of melanin. Tyrosinase inhibitors have gained interest in the cosmetics industry to prevent hyperpigmentation and skin-related disorders by inhibiting melanin production. It has been reported that several Aloe species exhibit anti-tyrosinase efficacy in vitro. In this study, the exudates of thirty-nine South African Aloe species were screened to identify species and compounds with anti-tyrosinase activity. Qualitative screening revealed that twenty-nine Aloe species exhibited tyrosinase inhibition activity with one to three active bands. Quantitative screening was performed for 29 species and expressed as IC50 values. Three species were further analysed and subsequently, aloesin and aloeresin A was isolated from A. ferox and plicataloside from A. plicatilis and A. chabaudii. Aloeresin A was determined to be a substrate of mushroom tyrosinase. Dose-response assays showed that aloesin (IC50 = 31.5 µM) and plicataloside (IC50 = 84.1 µM) exhibited moderate to weak activity. Molecular docking scores for plicataloside were considerably lower than for aloesin (P < 0.01), confirming its lower IC50. Several Aloe species may have potential for the management of hyperpigmentation or as a skin lightening agent. This is the first report showing that plicataloside, present in A. plicatilis and A. chabaudii, exhibits anti-tyrosinase activity.

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.

Screening selected medicinal plants for potential anxiolytic activity using an in vivo zebrafish model.

RATIONALE: Medicinal plants are used extensively in many countries to treat conditions related to the central nervous system (CNS), and there is renewed interest to explore natural products, which may exhibit CNS activity. OBJECTIVE: In this study, seven plants were selected based on literature reports of their ethnopharmacological use in treating anxiety-related conditions and assayed in a zebrafish model. METHODS: Crude extracts were prepared with solvents of different polarities, and the maximum tolerated concentration (MTC) of these crude extracts was established. The anxiolytic activity of the crude extracts was determined using 5-day post-fertilization (dpf) zebrafish larvae. General locomotor activity and reverse-thigmotaxis behavior (indicative of anxiolytic activity) were analyzed under continuous illumination and alternating light-dark challenges, which induced anxiety in the zebrafish larvae. RESULTS: Of the 28 extracts tested, 13 were toxic according to the MTC values obtained. Larvae were subsequently treated with the 15 non-toxic extracts, at a dose determined in the MTC assay or with 1% DMSO as control. The anxiolytic activity (reverse-thigmotaxis) was demonstrated by an increase in the percentage time spent by the larvae in the central arena of the well. Of the 15 non-toxic extracts tested, the Sceletium tortuosum water extract exhibited the most promising anxiolytic activity. CONCLUSIONS: The zebrafish model was effective in studying anxiety-related behavior. Thus, the study confirmed that S. tortuosum mitigates anxiety in zebrafish larvae, a step towards the full in vivo validation of the traditional use of the plant.