The effect of environmental factors on the metabolomic profile and the biological activities of Helichrysum aureonitens

Abstract

The use of plants for medicinal purposes precedes human written history. Accounts from many archaeological sources agree that the Sumerians were the first to compile plants’ lists according to their herbal remedy potentials over 5, 000 years ago. The World Health Organisation (WHO) estimated that about 80% of the world population rely on herbal medicine as their primary source of healthcare. There is a heightened public interest in the use of plant sources as natural therapies in both developed and developing countries in the last decade, leading to a surge in the availability of herbal remedies in pharmaceutical stores and supermarkets. The Helichrysum genus comprises flowering plants which are aromatic perennial shrubs belonging to the sunflower family. There are an estimated 600 species spread across Africa, Europe, Australasia, and Eurasia with almost half of the species found in South Africa. Helichrysum aureonitens is an important medicinal plant used for the treatment of many infections especially in the KwaZulu-Natal and Eastern Cape provinces. Previous studies on H. aureonitens have established antimicrobial activities against Gram positive bacteria (especially Bacillus species) and antiviral activity against viruses such as herpes simplex virus type 1 (HSV-1), coxsackie B virus type 1 (Cox B1), adenovirus type 31 (Ad31) and reovirus. Many phenolic compounds linked to the plants’ activities include chlorophenol, 4-chloro-2-(hepta-1,3,5-triyn-1-yl)-phenol, galangin (3,5,7 trihydroxyflavone) and chlorogenic acids among others have been identified from the shoot and leaves of H. aureonitens. Many studies have established the influence of environmental factors on plants’ secondary metabolites. Only a few studies however exist on the influence of seasonal variation or different growing locations with dissimilar climates on the chemical profile and antimicrobial activities of any plant species from the Helichrysum genus. This study therefore focused on determining the effect that change in seasons and locality with different climate has on the chemical profile of H. aureonitens and how the shift in metabolites is linked to the plants’ bioactivity. The antibacterial, antifungal, and antiviral activities of H. aureonitens extracts from different collection sites were evaluated against certain pathogens. The Gram-negative bacterium Proteus vulgaris and Gram-positive bacterium Bacillus subtilis were used for the study. The extracts’ activities were also tested against five pathogenic fungal species including Aspergillus flavus, Aspergillus nomius, Cladosporium cladosporioides, Fusarium oxysporum and Penicillum halotolerans. Lastly, the extracts were evaluated against the human virus, herpes simplex virus type 1 (HSV-1) to determine the antiviral activity. Plant samples were collected during the spring (October) and autumn (May) seasons at Wakefield farms, Midlands in KwaZulu-Natal (wetter location with lower temperature) and Telperion farm in Mpumalanga (drier location with higher temperature) regions of South Africa. Collections were made in both wet and dry sites selected at each of the two localities. Temperature and rainfall data were also collected for Cedara and Witbank between August 2017 and June 2018 representing the closest stations of South African Weather Services to the two collection locations. Hydroalcoholic extracts (30:70) of most H. aureonitens plants collected from both geographical locations and seasons showed good activities against the gram-negative bacterium P. vulgaris with Minimum Inhibitory Concentration (MIC) ranging between 62.5- 125 µg/mL. Activities of extracts collected from spring however showed better activities than the autumn harvests. No activity was however recorded against the gram-positive bacterium B. subtilis. Acetone extracts of H. aureonitens showed strong antifungal properties against four of the five fungal species tested. No activity was recorded against A. nomius by any of the extracts. All the other fungal species were however significantly inhibited by varied extract concentrations of H. aureonitens with MIC values between 0.39 – 3.125 µg/mL. The observed activities were specific to individual species. Generally, plant samples harvested from Telperion (drier location) had better activity against A. flavus, C. cladosporioides, F. oxysporum, and P. halotolerans compared to Wakefield (wet location). With the exception of F. oxysporum, rainfall had an inconsequential impact on the antifungal properties of the plant against the other tested species. A cytopathic effect (CPE) reduction approach was used to evaluate the anti-HSV activity of the twenty-six samples of H. aureonitens leaves and stems extracts. Cytotoxicity evaluation of the extracts was carried out using MTT assay prior to antiviral determination. It was observed that seventeen (mostly spring collections) of the twenty-six extracts examined were found to have considerable anti-HSV activity, as measured by a reduction in tissue culture infectious dose (TCID50) of less than 105. Through the application of multivariate statistical analysis using a Nuclear Magnetic Resonance spectrometer (NMR), 1H-NMR spectra of H. aureonitens leaf extracts were generated to compare samples collected in different seasons and locations as well as wet and dry sites. It was observed that changes in season played a significant role in the production of aromatics with the largest concentration found in the spring season and wet sites across both locations. Comparatively, the aromatics level is also favoured in the wetter geographical location which is Wakefield farm. Caffeoylquinic acids (CQA) which is the main Chlorogenic acids reported in nature are a class of compounds reported to be abundant in the Helichrysum genus. Ultra-Performance Liquid Chromatography Quadrupole Time of Flight Spectroscopy (UPLC-qTOF-MS), was used as an analytical platform to determine the identity of three derivatives of CQA and their isomers-monocaffeoylquinic acids (MCQA), dicaffeoylquinic acid (DCQA) and tricaffeoylquinic acid (TCQA) in the chemical profile of H. aureonitens. The DCQA derivative was the most abundant, with higher concentrations of all its isomers in both locations and seasons. The study highlights the impact of seasonal variation and different geographical locations on the chemical profile and antimicrobial properties of H. aureonitens.