The aim of the present study was to examine three different Galium species from the native population of Estonia, Galium verum, Galium aparine, and Galium mollugo, to characterise their non-volatile and volatile phytochemical composition and antioxidant activity. The main groups of bioactive compounds in the plants were quantified by colorimetric tests, showing high concentrations of polyphenols (up to 27.2 ± 1.5 mg GAE/g), flavonoids (up to 7.3 ± 0.5 mg QE/g) and iridoids (up to 40.8 ± 2.9 mg AE/g). The species were compared using HPLC-DAD-MS/MS, revealing some key differences in the phytochemical makeup of the extracts. The most abundant compound in the extracts of Galium verum blossoms and herb was found to be asperuloside, in Galium aparine herb, asperulosidic acid, and in Galium mollugo herb, chlorogenic acid. Additionally, the composition of volatile compounds was analysed by SPME-GC-MS. The degree of variability between the samples was high, but three volatiles, hexanal, anethole, and β-caryophyllene, were quantified (≥1%) in all analysed samples. The antioxidative activity of all extracts was evaluated using the ORAC_FL method, demonstrating that the Galium species from Estonia all exhibit strong antioxidant capacity (up to 9.3 ± 1.2 mg TE/g). Out of the extracts studied, Galium verum blossoms contained the highest amounts of bioactives and had the strongest antioxidant capacity.

Lyme disease, caused by Borrelia burgdorferi sensu lato infection, is the most widespread vector-borne illness in the Northern Hemisphere. Unfortunately, using targeted antibiotic therapy is often an ineffective cure. The antibiotic resistance and recurring symptoms of Lyme disease are associated with the formation of biofilm-like aggregates of B. burgdorferi. Plant extracts could provide an effective alternative solution as many of them exhibit antibacterial or biofilm inhibiting activities. This study demonstrates the therapeutic potential of Plantago major and Plantago lanceolata as B. burgdorferi inhibitors. Hydroalcoholic extracts from three different samples of each plant were first characterised based on their total concentrations of polyphenolics, flavonoids, iridoids, and antioxidant capacity. Both plants contained substantial amounts of named phytochemicals and showed considerable antioxidant properties. The major non-volatile constituents were then quantified using HPLC-DAD-MS analyses, and volatile constituents were quantified using HS-SPME-GC-MS. The most prevalent non-volatiles were found to be plantamajoside and acteoside, and the most prevalent volatiles were β-caryophyllene, D-limonene, and α-caryophyllene. The B. burgdorferi inhibiting activity of the extracts was tested on stationary-phase B. burgdorferi culture and its biofilm fraction. All extracts showed antibacterial activity, with the most effective lowering the residual bacterial viability down to 15%. Moreover, the extracts prepared from the leaves of each plant additionally demonstrated biofilm inhibiting properties, reducing its formation by 30%.

Lyme disease (LD) is a tick-borne bacterial disease that is caused by Borrelia burgdorferi. Although acute LD is treated with antibiotics, it can develop into relapsing chronic form caused by latent forms of B. burgdorferi. This leads to the search for phytochemicals against resistant LD. Therefore, this study aimed to evaluate the activity of Dipsacus fullonum L. leaves extract (DE) and its fractions against stationary phase B. burgdorferi in vitro. DE showed high activity against stationary phase B. burgdorferi (residual viability 19.8 ± 4.7%); however, it exhibited a noticeable cytotoxicity on NIH cells (viability 20.2 ± 5.2%). The iridoid-glycoside fraction showed a remarkable anti-Borrelia effect and reduced cytotoxicity. The iridoid-glycoside fraction was, therefore, further purified and showed to contain two main bioactives—sylvestrosides III and IV, that showed a considerable anti-Borrelia activity being the least toxic to murine fibroblast NIH/3T3 cells. Moreover, the concentration of sylvestrosides was about 15% of DE, endorsing the feasibility of purification of the compounds from D. fullonum L. leaves.

Borrelia burgdorferi sensu lato bacteria are the causative agent of Lyme disease, Europe’s most common vector-borne disease