Re: Phytochemical and Antibacterial Study of Helichrysum Extract

Helichrysum (Immortelle; Helichrysum italicum, Asteraceae) Lab Analysis Date: 01-15-2018 HC# 061762-584 Re: Phytochemical and Antibacterial Study of Helichrysum Extract D'Abrosca B, Buommino E, Caputo P, et al. Phytochemical study of Helichrysum italicum (Roth) G. Don: spectroscopic elucidation of unusual amino-phlorogucinols [sic] and antimicrobial assessment of secondary metabolites from medium-polar extract. Phytochemistry. December 2016;132:86-94. Several species of infectious bacteria, including Staphylococcus epidermidis and Pseudomonas aeruginosa, can form adherent layers on surfaces known as a biofilm. A biofilm formation may render antibiotics unable to attenuate infection. Helichrysum (Helichrysum italicum, Asteraceae) is used traditionally in combating inflammation and infection, and compounds such as phloroglucinols and their derivatives have been shown to have antibacterial activity. This basic research study isolated three novel amino-phloroglucinol-derivative compounds, and along with 17 other known compounds, tested their antimicrobial activity in assays measuring bacterial growth and biofilm inhibition. Plant material was collected in the Nature Reserve of Caserta, Italy, with a voucher stored in the herbarium at the Second University of Naples; Caserta, Italy. Leaves and stems were separated, dried, and extracted with solvent partitioning. Ultraviolet (UV) and nuclear magnetic resonance (NMR) spectroscopy, as well as thin-layer chromatography (TLC), were used to identify the compounds. Antimicrobial assays were done using S. epidermidis and Pseudomonas aeruginosa, with positive controls, tobramycin and vancomycin, to gauge the activity. Bacteria viability was measured as percent reduction in growth and growth inhibition after zero, four, eight, 16, and 24 hours of incubation with helichrysum extract dilutions. Biofilm impact was determined by incubating plant extracts with bacteria and measuring biofilm formation. Three novel amino-phloroglucinol derivatives and 17 previously reported compounds were isolated. In addition to phloroglucinols, compounds included acetophenone derivatives, with known compounds occurring in sandy everlasting (H. arenarium), imphepho (H. odoratissimum), many stem cudweed (Gnaphalium polycaulon, Asteraceae), shan you gan (Acronychia pedunculata, Rutaceae), Billy Webb (Acosmium panamense, Fabaceae), sour cherry (Prunus cerasus, Rosaceae), Cenostigma macrophyllum (Fabaceae), and lesser galangal (Alpinia officinarum, Zingiberaceae), among others. Four of the compounds, at 128 µg/ml, inhibited the growth of S. epidermidis at percentages ranging from 30.3 ± 0.03% to 77.0 ± 0.13%. In comparison, vancomycin was 100 ± 0.5% effective at the same concentration. These same compounds were tested for their time-kill efficacy against S. epidermidis. Compound 2, helichrytalicine B, demonstrated a better than 50% decrease in percentage of colony-forming units (CFU) after eight hours. Other compounds tested did not appreciably impact CFU despite inhibiting cell growth in the previous assay. In the biofilm assay, several compounds inhibited S. epidermidis biofilm formation, with compound 2 being the most efficacious (greater than 75% inhibition). Compound 9, an acetophenone derivative, and compound 20, ursolic acid, also were effective, approaching 50% inhibition. [Note: Exact percentages are not reported.] None of the compounds tested attenuated Pseudomonas aeruginosa viability. In conclusion, several novel, as well as known, compounds were successfully isolated from helichrysum. The authors mention that variations between compounds in antimicrobial mechanisms may reflect compound structural differences, as well as efficacy for either Gram-negative or Gram-positive bacteria. The data reported here suggest compound 2 as the most efficacious in all three antimicrobial assays. It is posited that compounds effective at both growth and biofilm inhibition may be worthy of further investigation as antibacterial therapies. One of the authors (Scognamiglio) acknowledged support from the L'Oréal UNESCO (United Nations Educational, Scientific and Cultural Organization) program "For Women in Science" through the 2012 National Fellowship "L'Oréal Italia per le Donne e la Scienza." —Amy C. Keller, PhD