Identification of Anti-HIV Biomarkers of Helichrysum Species by NMR-Based Metabolomic Analysis.

Several species of the Helichrysum genus have been used ethnobotanically to treat conditions that we today know have been caused by viral infections. Since HIV is a modern disease with no ethnobotanical history, we commenced with a study on the anti-HIV activity of several Helichrysum species. Drug discovery of small molecules from natural resources that is based on the integration of chemical and biological activity by means of metabolomical analyses, enables faster and a more cost-effective path to identify active compounds without the need for a long process of bioassay-guided fractionation. This study used metabolomics to identify anti-HIV compounds as biomarkers from 57 Helichrysum species in a combined study of the chemical and biological data of two previous studies. In the OPLS-DA and hierarchical cluster analyses, anti-HIV activity data was included as a secondary observation, which assisted in the correlation of the phytochemical composition and biological activity of the samples. Clear grouping revealed similarity in chemical composition and bioactivity of the samples. Based on the biological activity of polar extracts, there was a distinct phytochemical difference between active and non-active groups of extracts. This NMR-based metabolomic investigation showed that the chlorogenic acids, compounds with cinnamoyl functional groups, and quinic acid were the most prominent compounds in the Helichrysum species with anti-HIV activity. This study further revealed that the chlorogenic acid type compounds and quinic acid are biomarkers for anti-HIV activity.

Isolation and identification of a novel chlorophenol from a cell suspension culture of Helichrysum aureonitens.

A novel chlorophenol, 4-chloro-2-(hepta-1,3,5-triyn-1-yl)-phenol (1), was isolated as the major phenolic compound from the cells of Helichrysum aureonitens suspension cultures. Compound 1 has been proposed to be an intermediate in the acetylene biosynthetic pathway of other acetylenic compounds in Helichrysum spp. The ethanol extract of cell suspension cultures and compound 1 were evaluated for their cytotoxicity against monkey kidney Vero (Vero cells) and human prostate epithelial carcinoma (DU145) cell lines, also, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Mycobacterium tuberculosis H37Rv were determined as well.

Identification of anti-HIV active dicaffeoylquinic- and tricaffeoylquinic acids in Helichrysum populifolium by NMR-based metabolomic guided fractionation.

South Africa being home to more than 35% of the world's Helichrysum species (c.a. 244) of which many are used in traditional medicine, is seen potentially as a significant resource in the search of new anti-HIV chemical entities. It was established that five of the 30 Helichrysum species selected for this study had significant anti-HIV activity ranging between 12 and 21 µg/mL (IC50) by using an in-house developed DeCIPhR method on a full virus model. Subsequent toxicity tests also revealed little or no toxicity for these active extracts. With the use of NMR-based metabolomics, the search for common chemical characteristics within the plant extract was conducted, which resulted in specific chemical shift areas identified that could be linked to the anti-HIV activity of the extracts. The NMR chemical shifts associated with the activity were identified to be 2.56-3.08 ppm, 5.24-6.28 ppm, 6.44-7.04 ppm and 7.24-8.04 ppm. This activity profile was then used to guide the fractionation process by narrowing down and focusing the fractionation and purification processes to speed up the putative identification of five compounds with anti-HIV activity in the most active species, Helichrysum populifolium. The anti-HIV compounds identified for the first time from H. populifolium were three dicaffeoylquinic acid derivatives, i.e. 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid as well as two tricaffeoylquinic acid derivatives i.e. 1,3,5-tricaffeoylquinic acid and either 5-malonyl-1,3,4-tricaffeoylquinic or 3-malonyl-1,4,5-tricaffeoylquinic acid, with the latter being identified for the first time in the genus.

Comparative functional analysis of CYP71AV1 natural variants reveals an important residue for the successive oxidation of amorpha-4,11-diene.

Artemisinin is an antimalarial sesquiterpenoid isolated from the aerial parts of the plant Artemisia annua. CYP71AV1, a cytochrome P450 monooxygenase was identified in the artemisinin biosynthetic pathway. CYP71AV1 catalyzes three successive oxidation steps at the C12 position of amorpha-4,11-diene to produce artemisinic acid. In this study, we isolated putative CYP71AV1 orthologs in different species of Artemisia. Comparative functional analysis of CYP71AV1 and its putative orthologs, together with homology modeling, enabled us to identify an amino acid residue (Ser479) critical for the second oxidation reaction catalyzed by CYP71AV1. Our results clearly show that a comparative study of natural variants is useful to investigate the structure-function relationships of CYP71AV1.

Purified compounds and extracts from Euclea species with antimycobacterial activity against Mycobacterium bovis and fast-growing mycobacteria.

Naphthoquinones and other compounds with antimycobacterial activity against Mycobacterium tuberculosis have previously been isolated from Euclea species. In this study, several constituents of Euclea natalensis and E. undulata, as well as organic extracts of the leaves, were assessed for efficacy against the zoonotic pathogen, Mycobacterium bovis. Also included in the battery of test organisms were M. bovis BCG and the fast-growing species M. smegmatis and M. fortuitum. The acetone extract of E. natalensis had potent activity against M. bovis (MIC=26 microg/ml). The naphthoquinone 7-methyljuglone was the most active compound, with an MIC as low as 1.55 microg/ml against pathogenic M. bovis. M. bovis BCG was not as susceptible to the test compounds as the pathogenic strain, but similar patterns of activity were observed between all the strains tested. M. smegmatis appeared to be a better predictor of antimycobacterial activity against pathogenic M. bovis (and M. tuberculosis), while MIC values obtained using M. fortuitum correlated well with those of M. bovis BCG.