Elucidation Of Multi-origin Inula Helenium L. For Antimicrobial Activity And Heavy Metal Profiling As An Exemplar For The Use And Regulation Of Plants As Antibiotics.
Authors:   ,   Munster Technological University, Kenny Ciara 
Published: August 2024

An impending post-antibiotic era looms, thus new sources of therapeutic chemical entities are crucial to sustain effective antimicrobial chemotherapy worldwide. Plant natural products are a source of accessible, structurally diverse compounds that provide therapeutic potential. The pharmacological applications of plants in medicine can be guided by the attestation of traditional use or the application of this knowledge to uncover new leads in the drug discovery process. The range of modified in vitro methods commonly used to investigate preclinical antimicrobial efficacy of plant-derived natural products, and the associated limitations and challenges in the provision of new antimicrobial drugs from plants is discussed. Currently, there are no approved guidelines, standards, or official recommendations governing in vitro antimicrobial methodologies for natural products of plant origin thus jeopardising transparency within this field of research. Standardisation of methodology will support future in vitro R&D and efficacy testing of plant-derived antibiotics. Evidence shows that the rate of new antimicrobial development is insufficient to meet our current and future needs globally. This, along with a weak drug portfolio pipeline, emphasises the importance – and relevance - of investigating prosperous sources of potentially new structures, such as plants. Inula helenium L. (elecampane) is a perennial herb of the Asteraceae family, naturalised in Ireland. Irish ethnobotanical indications centre around respiratory, digestive, and dermal ailments. A key research objective of this project was to identify the natural product compound(s) attributing to the anti-staphylococcal activity of a traditional hydroethanolic extract of multi-origin elecampane root, previously observed within our laboratory. This thesis explores the application of a novel clean-up strategy and bioactivity-guided fractionation process which resulted in a subset of antibacterial or ‘active’ fractions. The phytochemical composition of these fractions was later analysed using a validated high-performance liquid-chromatography with diode array detection (HPLC-DAD) method supported by 1H nuclear magnetic resonance (NMR). The natural products attributing to the observed in vitro activity were identified as alantolactone, isoalantolactone, igalan(e), and an unseparated mixture of dugesialactone and alloalantolactone as major constituents. Another finding of this study was that the geographical origin of elecampane did not appear to influence either the chemical profile xix or the bioactivity of the analysed traditional root extracts. Elecampane clearly demonstrates activity against Staphylococcus spp. and considering the occurrence of antimicrobial resistance in Irish hospitals among this genus and the high prevalence of MRSA, further investigation is warranted. This body of work therefore justifies elecampane as a promising reservoir of antimicrobial compounds. Follow-on studies could include large-scale purification or synthesis of the identified compounds followed by in vivo analysis of the compounds, individually and in combination, and combinatorial experimentation as potentiator or adjuvant compounds to support conventional antibiotic treatment. The concept of elemental impurification of plant material, the consequences of exposure to human health, and the regulations that are currently in place to safeguard the general European population, are explored in this thesis. Plants acquire metals from the environment during cultivation and collection (geogenic and anthropogenic-derived sources), and as elemental impurities during processing and manufacturing. Toxicologically significant metals can thus arise as unsolicited and hazardous residues in plants and products thereof, thus plants have been shown to be a risk factor for human exposure to metals. While regulatory measures are enforced in European Member States, there are apparent gaps in legislation concerning the presence of certain metals in plantderived food and phytopharmaceuticals. The adapted ICH Q3D guidelines published by the EMA define limits for a total of twenty-four elements, however, these limits are not currently applicable to herbs. In contrast, compendial (Ph. Eur.) limits for herbal products exist for only Cd, Hg and Pb. From reviewing the literature, our investigation suggests that two possible solutions to consider include extending the application of ICH Q3D guidelines to cover herbal products, or alternatively, the establishment of a defined set of general permissible limits for a greater suite of toxicologically significant metals applicable to herbal products. Levels within plant-derived food and medicinal products are not always routinely analysed, nor are patients’ samples routinely tested for metal levels in clinical settings. Trace element interactions can influence nutritional status and interfere with normal biological functions in humans. It is difficult to know the extent that metal contaminants play in the symptoms or aetiology of chronic human disease, therefore, harmonisation of quality requirements for food and medicinal products is a necessity, particularly in the context of international trade and assurance of consumer safety. xx Two investigations were performed to determine the multi-elemental (metallomic) profiles of elecampane (n = 27) and other commonly-used medicinal plant species (n = 50), using a validated high-resolution inductively coupled plasma sector-field mass spectrometry (HR-ICP-SFMS) method following microwave acid-digestion. For the elecampane study, the research objective was to quantify for the first time, the novel multi-elemental (metallomic) profile of naturalised Irish and commercial samples of elecampane and investigate the risk of dietary exposure in silico using the EFSA RACE tool to contextualise the toxicological significance of acute and/or chronic dietary exposure to metal contaminants. Results from the analysis showed that chronic exposure to lead (Pb) at a maximum quantified concentration of 4617.42 μg.kg-1 is of potential risk to adult consumers in Ireland (18-65 y), at an estimated mean and 95th percentile exposure of 0.049 and 0.189 μg⋅(kg BW)−1d−1, respectively. Further investigation is advised, including soil and water analysis from the cultivation land to ascertain if Pb levels are within acceptable environmental limits, particularly if the farms are producing herbs, vegetables or crops for local communities. Additionally, 52% of the elecampane samples were found to exceed European limits for Cd in food (200 μg.kg-1), and one sample exceeded the compendial ML for Cd impurities in herbal material/drugs (> 1 mg.kg-1). These findings illustrate the availability of non-compliant herbal substances, purchasable by local communities and online consumers. Dietary exposure to Cd at the highest observed concentration (1285.97 μg.kg-1) however, despite exceeding European regulatory and pharmacopeial permissible limits, was deemed “no risk” to Irish consumers when considering consumption of this single food category. The remaining elements (Li, Be, Mo, Sn, Ba, Hg, Tl, V, Cr, Co, Ni, Cu) were well below regulatory and pharmacopeial limits in all samples and thus dietary exposures to these elements are therefore of negligible concern to Irish consumers. Outputs from the RACE tool, however, focus on one contaminant in one food commodity at a time, and therefore background exposure from all other dietary sources needs consideration. Another major finding of the elecampane investigation revealed a significant difference in element distribution between flowers – leaves (Be, Li, Ba, Cd, Bi), root – leaves (Mo, Sn, Co, Hg) and flowers – root (Ba and Tl) in naturalised elecampane samples. Considering the range of elements tested, the geographical distribution of the sampled plants and the sufficient sample size, the findings of this investigation can be used in future studies as a control population for inter-study comparisons. The multi-elemental (metallomic) profiles of fifty medicinal plant samples was investigated, many for the first-time, including arnica, bush vetch, sweet cicely, yellow rattle, bogbean, rock-tea and tufted catchfly. Highest quantified concentrations were observed in ox-eye daisy flower (Li: 3964 μg.kg-1), dandelion leaf (Be: 122 μg.kg-1; Cd: 325 μg.kg-1; and Sn: 165 μg.kg-1), dandelion root (Ti: 5827 μg.kg-1), great mullein leaf (Mo: 4505 μg.kg-1; and Tl: 91 μg.kg-1), elderberry fruit (Ba: 4646 μg.kg-1), hawthorn flower and leaf (Pt: (33 μg.kg-1); Hg: (30 μg.kg-1), Pb: 4248 μg.kg-1; and Bi: 30 μg.kg- 1), comfrey root (V: 1758 μg.kg-1), boldo leaf (Cr: 4534 μg.kg-1), coltsfoot leaf (Co: 652 μg.kg-1) and flower (Ni: 6060 μg.kg-1), and lastly aerial bush vetch (Cu: 6340 μg.kg-1). Shorter-than-lifetime daily intakes were estimated using conservative (“worst-case”) and realistic theoretical exposure scenarios. The non-carcinogenic risk assessment was subsequently evaluated using the Hazard Quotient (HQ) and Hazard Index (HI) mathematical equations. All botanical samples analysed (n = 50) were below the compendial limits for metal impurities in herbal substances/starting materials: Cd (≤ 1 mg.kg-1), Hg (0.1 mg.kg-1), and Pb (5 mg.kg-1), however, Li, Mo, Tl, Pb, Co, and Ni were quantified at potentially unsafe levels at the theoretical worst-case exposure scenario. . Furthermore, 42% of all samples tested, representing 16 different plant species (n = 30), were categorised as potentially unsafe to consumers (HI ≥ 1) with regards to the noncarcinogenic cumulative exposure to Cu, Cd, Hg and Pb, including: hawthorn, arnica, dandelion, marigold, nettle, yarrow, comfrey, borage, coltsfoot, birds foot trefoil, ox-eye daisy, yellow rattle, mugwort, great mullein, tufted catchfly, marshmallow. Major data gaps hindering the risk evaluation of plants and derived products are discussed. Intake and consumption data at European level and the availability of guidance in the absence of experimental data (e.g. metal transfer rates, exposure durations and frequency) to facilitate harmonised deterministic and probabilistic risk assessment criteria would help ensure botanical safety. In conclusion. the work presented in this thesis aims to provide a basis for plant research methodology standardisation, and harmonisation or extension of inclusion criteria within pharmacognosy and the wider botanical sciences to assure botanical safety, quality and regulation – from initial screening to product commercialisation.</p>