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

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

Resprouters Versus Reseeders: Are Wild Rooibos Ecotypes Genetically Distinct?

Aspalathus linearis (Burm. F.) R. Dahlgren (Fabaceae) or rooibos, is a strict endemic species, limited to areas of the Cederberg (Western Cape) and the southern Bokkeveld plateau (Northern Cape) in the greater Cape Floristic Region (CFR) of South Africa. Wild rooibos, unlike the cultivated type, is variable in morphology, biochemistry, ecology and genetics, and these ecotypes are broadly distinguished into two main groups, namely, reseeders and resprouters, based on their fire-survival strategy. No previous assessment of genetic diversity or population structure using microsatellite markers has been conducted in A. linearis. This study aimed to test the hypothesis that wild rooibos ecotypes are distinct in genetic variability and that the ecotypes found in the Northern Cape are differentiated from those in the Cederberg that may be linked to a fire-survival strategy as well as distinct morphological and phytochemical differences. A phylogeographical and population genetic analyses of both chloroplast (trnLF intergenic region) and newly developed species-specific nuclear markers (microsatellites) was performed on six geographically representative wild rooibos populations. From the diversity indices, it was evident that the wild rooibos populations have low-to-moderate genetic diversity (He: 0.618-0.723; Ho: 0.528-0.704). The Jamaka population (Cederberg, Western Cape) had the lowest haplotype diversity (H = 0.286), and the lowest nucleotide diversity (π = 0.006) even though the data revealed large variations in haplotype diversity (h = 0.286-0.900) and nucleotide diversity (π = 0.006-0.025) between populations and amongst regions where wild rooibos populations are found. Our data suggests that populations of rooibos become less diverse from the Melkkraal population (Suid Bokkeveld, Northern Cape) down towards the Cederberg (Western Cape) populations, possibly indicative of clinal variation. The largest genetic differentiation was between Heuningvlei (Cederberg, Western Cape) and Jamaka (FST = 0.101) localities within the Cederberg mountainous region, and, Blomfontein (Northern Cape) and Jamaka (Cederberg) (FST = 0.101). There was also a significant isolation by distance (R2 = 0.296, p = 0.044). The presence of three main clusters is also clearly reflected in the discriminant analysis of principal components (DAPC) based on the microsatellite marker analyses. The correct and appropriate management of wild genetic resources of the species is urgently needed, considering that the wild Cederberg populations are genetically distinct from the wild Northern Cape plants and are delineated in accordance with ecological functional traits of reseeding or resprouting, respectively. The haplotype divergence of the ecotypes has also provided insights into the genetic history of these populations and highlighted the need for the establishment of appropriate conservation strategies for the protection of wild ecotypes.

Antimicrobial interactions of Khoi-San poly-herbal remedies with emphasis on the combination; Agathosma crenulata, Dodonaea viscosa and Eucalyptus globulus.

ETHNOPHARMACOLOGICAL RELEVANCE: Plants are often combined in traditional herbal remedies to increase medicinal efficacy, thus this investigation provides some insight into the antimicrobial efficacies of selected combinations. AIMS OF THE STUDY: The first aim was to scientifically validate antibacterial efficacy of plant mixtures that are traded within peri-urban centres of Cape Town (Western Cape, South Africa). This was followed by an in-depth evaluation of the most antimicrobially active mixture; Agathosma crenulata, Dodonaea viscosa with Eucalyptus globulus. MATERIALS AND METHODS: Methanol and aqueous extracts of six plant mixtures were screened for antibacterial properties against two Gram-negative and two Gram-positive bacteria using the minimum inhibitory microdilution method. Thereafter, chloroform: methanol (1:1; v/v) extracts, essential oils and aqueous extracts of Agathosma crenulata, Dodonaea viscosa and Eucalyptus globulus were assayed for antimicrobial activity independently and in various combinations. The fractional inhibitory concentration indices (Æ©FIC) were determined for double and triple plant combinations to establish antimicrobial interactions. RESULTS: From the six plant mixtures prepared by herbalists, a methanol extract derived from combining Agathosma crenulata, Dodonaea viscosa and Eucalyptus globulus showed the best antibacterial activity. The MIC values of 49µg/ml for Staphylococcus aureus and Bacillus subtilis, and 98µg/ml for Klebsiella pneumoniae and Escherichia coli were recorded. When Agathosma crenulata, Dodonaea viscosa and Eucalyptus globulus were mixed in various 1:1 combinations, mostly additive and synergistic interactions were noted. The most noteworthy synergistic (ΣFIC value 0.07) 1:1 combinations were observed for the chloroform: methanol extracts of Agathosma crenulata mixed with Eucalyptus globulus against Klebsiella pneumoniae, Staphylococcus aureus and Bacillus subtilis. When combined in a mixture of three plants (1:1:1), enhanced efficacy was evident against most of the pathogens, for both organic and aqueous extracts. The triple combination against Bacillus subtilis demonstrated the greatest synergy (ΣFIC values of 0.03). CONCLUSIONS: The enhanced antimicrobial efficacy and synergistic interactions noted for some of the mixtures, particularly the combination of Agathosma crenulata, Dodonaea viscosa and Eucalyptus globulus support the Western Cape Khoi-San traditional medicinal practices of combining plants for enhanced efficacy.

Genetic alterations for increased coumarin production lead to metabolic changes in the medicinally important Pelargonium sidoides DC (Geraniaceae).

The medicinal plant Pelargonium sidoides is fast becoming threatened due to the overharvest of its tubers from the wild to produce a phytopharmaceutical for treating respiratory infections. The action of the coumarins is implicated in the efficacy of the commercial herbal extract with the highly oxygenated coumarins exhibiting the best anti-bacterial and anti-viral activity. Through this work we aimed at exploring the metabolic effects of Agrobacterium rhizogenes transformation. After confirmation of transgenesis using PCR amplification of the rol A (320 bp), rol B (400 bp) and rol C (600 bp) genes, metabolite profiles indicated a high level of variability between the different transgenic clones but these had more compounds compared to non-transgenic control cultures. This was represented by a two- to four-fold increase in detected metabolites in transgenic clones. We quantified several commercially important coumarins, flavonoids and phenolic acids. One of the clones had six out of nine of these metabolites. Overall, the concentration of these metabolites of interest were significantly changed in transgenic root cultures, for instance shikimic acid was recorded at the highest level in clone A4T-A. Production of key metabolites at significantly higher concentrations due to transgenesis and positive anti-bacterial activity exhibited by transgenic roots lends support to the idea of developing these clones as an alternative source that will allow for sustainable access to economically valuable secondary compounds of P. sidoides.

Current perspectives on an emerging formal natural products sector in South Africa.

Indigenous medical plants contribute significantly to a large South African population as part of a long-standing healthcare system intimately linked to folklore and for livelihood security. For the economically marginalized, access to such plants is largely through herbal markets which are part of an informal economy. Otherwise the formal natural products economy services those with a better socio-economic standing. Recently, the latter has experienced tremendous growth which largely mirrors the global cultural trend for organic naturopathies. Commercialisation of traditional plants and their contribution to the cosmeceutical, nutraceutical and pharmaceutical industries locally and abroad is reviewed. Traditional plant knowledge of southern African people is a source of inspiration for new product development. Concomitantly, an upsurge in research activities emanating from South Africa which confirms the pharmacological efficacy of these plants is fuelling a greater trust in indigenous flora. The escalating consumption of ethnomedicinals as highly valued commodities not only presents South Africa with socio-economic opportunities but also with challenges. Sustainable utilization benefiting the commodification of ethnoherbals, plus meeting aims of poverty alleviation and people empowerment, is a new paradigm in South Africa. The future sustainability of local ecosystems depends upon scientific conservation management practices that recognize the importance of involving local communities. Conservationists should remain aware and sensitive of socio-cultural dynamics within communities in order to manage natural resources.

South African Salvia species: a review of biological activities and phytochemistry.

The genus Salvia (sage) belongs to the Lamiaceae and encompasses 900 species worldwide of which ca. 26 indigenous species are found in southern Africa. Salvia is the largest genus in this family and constitutes almost one quarter of the Lamiaceae. In South Africa, the majority of Salvia species are distributed predominantly in the Cape region. Salvia species are used in many parts of the world to treat various conditions. Many sages, if not all, form an integral part of traditional healing in South Africa particularly in regions where they occur in abundance. Several species are used to treat microbial infections, cancer, malaria, inflammation, loss of memory and to disinfect homes after sickness. Despite the extensive traditional use and the general interest in phytoconstituents of Salvia it remains ironic that research on the South African counterparts has until recently been neglected. The review aims to collate recent research results on the phytochemistry and pharmacological properties of indigenous species. Bio-active compounds with antiplasmodial and antibacterial activities have been isolated and structurally elucidated from Salvia chamelaegnea, Salvia radula and Salvia verbenaca. The essential oil composition of Salvia showed the dominance of monoterpene hydrocarbons, oxygen-containing monoterpenes and oxygen- containing sesquiterpenes. Salvia runcinata is identified as an alternative source of natural alpha-bisabolol. Many pharmacological activities are summarised (anti-oxidant, antimicrobial, antiplasmodial, analgesic, antipyretic, anticancer, anti-inflammatory and antinociceptive) as a first attempt to provide scientific support for past and present local traditional uses.

Micropropagation of Thapsia garganica--a medicinal plant.

The micropropagation of T. garganica, a medicinally important plant, was investigated as an option for conservation purposes as wild populations are becoming sparse. A Murashige and Skoog agar medium supplemented with 0.5 mg l(-1) alpha-naphthaleneacetic acid and 1.5 mg l(-1) benzyl-6-adenine significantly improved the production of multiple shoots directly from petiole and leaflet explants compared to other plant growth regulator (PGR) combinations. Medium free of PGRs promoted rooting at a low incidence. Acclimatisation was low--52% plantlet survival--as micropropagated plantlets were highly susceptible to fungal rot once removed from culture. Application of antifungal agents to in vitro-derived plantlets as pre- and post-acclimatisation treatments during transplanting significantly reduced ex vitro mortality.