IllustratIon of the relevance of intraspecific variation driven by spatial isolation, seasonal differences, and their interaction in the medicinal value of plant : Helichrysum kraussii Sch. Bip. case study

Quantitative and qualitative differences in secondary metabolite profiles at the inter- and intraspecies levels determine the medicinal value of plants. Intraspecies variation is driven by genetic disparities and differences in abiotic and biotic stressors that different populations of a species may be exposed to. The importance of these differences is often overlooked, compromising the robustness of inferences regarding a species’ medicinal value. Ethanol extracts were prepared from 93 samples collected from nine populations and two seasons and analysed using high-performance thin-layer chromatography to elucidate secondary metabolite profiles. Select extracts were tested against Cutibacterium acnes to determine their half-maximal inhibitory concentration (IC50) values and evaluate whether significant differences in antibacterial activity were present between the samples. Fresh plant material was used in flow cytometry to determine the relative genome sizes of select populations using Pisum sativum L. and Helianthus annuus L. as the internal standards. Principal component analysis (PCA) and agglomerative hierarchical clustering (AHC) with bootstrap did not discernibly group samples based on their provenance or season of collection. Permutational analysis of variance using population, season, and their interaction, however, indicated that these factors significantly (p < 0.05) influenced variation in their secondary metabolite profiles. A k-means analysis grouped samples into two robust clusters, validated by a partial least squares discriminant analysis (5-fold validation, Q2 = 0.6, and permutation analysis, p < 0.01). Poor correlation between geographic distance and secondary metabolite profiles (R2 = 0.185, p = 0.263) was evident with a Mantel test.

Literature on the chemical makeup of H. kraussii reported the presence of kaurenoic acid (KA), a secondary metabolite found within several plant families with antibacterial activity. The compound was present in all samples, however, two-way analysis of variance indicated that concentrations differed significantly (p < 0.05) between five populations and seasons. The IC50 values of these samples ranged between 17.10 ± 4.16 μg/mL and 391.16 ± 15.02 μg/mL, whereas KA exhibited potent (IC50 = 37.94 ± 2.73 μg/mL) antibacterial activity. However, a linear regression model revealed that a poor correlation existed between the samples’ antibacterial activity and KA concentrations, suggesting that an unidentified compound was probably responsible for inhibiting the growth of C. acnes. A Kruskal-Wallis test indicated significant variation in antibacterial activity (Bonferroni corrected p < 0.0005) between samples. Finally, flow cytometry revealed that the Mountain Sanctuary Park population contained individuals with significantly (p < 0.05) smaller relative genomes. The Mantel test using the components of the PLS-DA and the genome sizes did not indicate that a correlation existed between these variables. This study concluded that season, population, and their interaction, drive interpopulation variation in secondary metabolite profiles in Helichrysum kraussii, however, because of the difficulty in identifying patterns within the clustering of sample along population and season, it is hypothesised that two chemotypes, that do not solely rely on these variables, of H. kraussii exist.