The plant kingdom contributes significantly to the natural products that are used for the treatment of a large number of ailments and disease across the globe. Included in these species is the Helichrysum genus (Asteraceae), which comprises of more then 600 species across Africa of which 244 species are found in South Africa. Helichrysum species are used in many cases for the treatment of coughs, colds, fever, infection, headaches, menstrual pain and are also very popular for wound dressing due to their potential antibacterial properties. The most common Helichrysum species used in traditional medicine and for several medicinal purposes are H. cymosum, H. odoratissimum, H. petiolare and H. nudifolium. Previously published research has shown that several of the Helichrysum species do have antimicrobial activity with the most relevant to this study being the discovery of antiviral activity of H. aureonitens against herpes simplex virus type 1 (HSV-1) as well as the reports of anti-HIV (human immunodeficiency virus) activity of several Helichrysum species. With this knowledge, a more in-depth study was initiated to identify the possible active constituents in South African Helichrysum species against HIV. Due to the need to speed up drug discovery especially against epidemic diseases like HIV, this study investigated a new tool (nuclear magnetic resonance (NMR) – based metabolomics) to speed up drug discovery form natural products especially when anti-viral constituents are investigated. odoratissimum, H. petiolare and H. nudifolium. Previously published research has shown that several of the Helichrysum species do have antimicrobial activity with the most relevant to this study being the discovery of antiviral activity of H. aureonitens against herpes simplex virus type 1 (HSV-1) as well as the reports of anti-HIV (human immunodeficiency virus) activity of several Helichrysum species. With this knowledge, a more in-depth study was initiated to identify the possible active constituents in South African Helichrysum species against HIV. Due to the need to speed up drug discovery especially against epidemic diseases like HIV, this study investigated a new tool (nuclear magnetic resonance (NMR) – based metabolomics) to speed up drug discovery form natural products especially when anti-viral constituents are investigated. In this study very promising anti-HIV results were obtained from several aqueous extracts (1:1 methanol/water) using a full virus model i.e. Helichrysum populifolium (IC50 12 μg/ml), H. appendiculatum (IC50 17 μg/ml), H. cymosum ssp. clavum (IC50 19 μg/ml), H. oxyphyllum (IC50 19 μg/ml) and H. cymosum ssp. cymosum (IC50 21 μg/ml). With the use of NMR-based metabolomics and multivariate data analysis (MVA) the specific characteristic that differentiated the active extracts from the non-active extracts was identified by making use of Orthogonal Projections to Latent Structures – Discriminant Analysis (OPLS-DA). This characteristic was then used as a “blue print” or “fingerprint” to guide the process of fractionation and purification. H. populifolium showed the highest anti-HIV activity and thus was selected as the candidate extract for further analysis. After a very quick and simple chromatographic fractionation process, seven fractions were compared against the activity profile by making use of their NMR profiles, which then visually indicated which of the fractions had the highest similarity. Fraction 6 had the most similar “fingerprint”. The compounds of this active fraction were then identified with the use of liquid chromatography – ion trap – time of flight (LC-IT-TOF) for quick identification. The analysis revealed the presence of five chlorogenic type compounds, 3,4-dicaffeoyl quinic acid (DCQA), 3,5-DCQA, 4,5-DCQA, 1,3,5- tricaffeoyl quinic acid (TCQA) and 5-malonyl-1,3,4-TCQA of which several are well known to have anti-HIV activity ranging from 0.85μM to 12μM. We were thus able to show with this study the possibility of using NMR-based metabolomics guided fractionation to guide the process of fractionation and identification from an active characteristic profile to the active constituents within the active H. populifolium extract. / Thesis (PhD)--University of Pretoria, 2013. / gm2014 / Plant Science / unrestricted

From the Helichrysum genus 600 species occur in Africa of which 244 species are found in South Africa. The most commonly used Helichrysum species for medicinal purposes are H. cymosum, H. odoratissimum, H. petiolare and H. nudifolium. The medicinal uses include the treatment of coughs, colds, fever, infection, headaches, menstrual pain and are very popular for wound dressing. Previous published research has shown that H. aureonitens has antiviral properties against Herpes simplex virus type 1 (HSV-1). In this study, further investigation into the Helichrysum species was undertaken, to establish the active constituents responsible for anti-HSV activity using a metabolomics approach. The cytotoxicity of 12 Helichrysum species was investigated and ranged from <3.125 μg/ml to 277.8 μg/ml on the vero cell line. The 12 Helichrysum species also showed various levels of antiviral activity against HSV, with both the water-methanol and chloroform extracts of H. adenocarpum subsp. adenocarpum being the most active extract at 25 μg/ml. In this study the activity of Helichrysum species against HIV-1 RT was also investigated. Helichrysum populifolium was the most active extract, inhibiting the HIV-1 RT enzyme by 63.78 % at 200 μg/ml. The bioactivity data and the spectral nuclear magnetic resonance (NMR) data of al the Helichrysum species from this study was analysed using the SIMCA-P software to discriminate between the different species on the basis of their bioactivity and chemical composition. The samples did not group well on Principal Component Analysis (PCA) but did separate well using the Orthogonal Projection to Latent Structure – Discriminate Analysis (OPLS-DA) on the basis of their activity and NMR spectra data. From the OPLS scoring plots analysis, contribution plots were created which indicated regions responsible for the difference between the species, with these regions being investigated to identify the bioactive constituents. It was thus possible to use metabolomics to discriminate between samples on the basis of their activity and show that it could probably be used in future as a tool to identify active ingredients in medicinal plants and accelerate drug discovery. Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Plant Science / unrestricted