# Phytochemical Screening and Antioxidant Activity of Selected Estonian _Galium_ Species
## 4 Conclusions  
This paper describes the phytochemical studies of three _Galium_ species native to northern Europe. Our results show that the selected Estonian _Galium_ species (_Galium vermum_ blossoms and herb, _Galium aparine_ herb, and _Galium mollugo_ herb) are a valuable source of phenolics with antioxidant properties, iridoids, and volatile phytochemicals. The quantitative studies showed that the most abundant non-volatile compound in the extracts of _Galium vermum_ blossoms and herb was asperuloside, in _Galium aparine_ herb, asperulosidic acid, and in _Galium mollugo_ herb, chlorogenic acid. The volatile compounds had a high degree of variability, but three, hexanal, anethole, and \(\beta\)-caryophyllene, were quantified (\(>\)1.0%) in all analysed samples. Additionally, it was found that some key differences in the chromatographic profiles of the extracts allow for a quick identification of an Estonian _Galium_ plant of an unknown species. The _Galium vermum_ extracts exhibited a comparatively large ruin peak, whereas _Galium aparine_ an enhanced cryptochlorogenic acid peak. The prominent peaks of diosmetin isomers set apart the _Galium mollugo_ extract from the others. The effects of the polarity of an extraction solvent were characterised by the fact that extraction with 50% acetone yielded extracts rich in polyphenols (up to 25.1 \(\pm\) 0.8 mg GAE/g) and flavonoids (up to 7.3 \(\pm\) 0.5 mg QE/g), whereas 50% ethanol was preferable for iridoid extraction (up to 36.6 \(\pm\) 2.2 mg AE/g). Both 50% ethanol and 50% acetone extracts were characterised by high antioxidant activities (up to 7.2 \(\pm\) 1.4 and 9.3 \(\pm\) 1.2 mg TE/g, respectively). As oxidative stress can lead to a variety of severe illnesses, the antioxidative properties of the _Galium_ extracts contribute to their potential as future therapeutic agents. Out of the species studied, the _Galium vermum_ blossoms had the strongest antioxidant properties (up to 9.3 \(\pm\) 1.2 mg TE/g). The therapeutic potential of several volatile and non-volatile phytochemicals found in the _Galium_ species has already been demonstrated. _Galium vermum_ is rich in asperuloside, which has been shown to have anti-viral, anti-malarial, anti-protozoan, anti-tumorigenic, anti-hypertensive, anti-obesity, immunomodulatory, anti-inflammatory, and antioxidant properties. Estonian _Galium_ species are rich in polyphenics, mainly chlorogenic acid and ruin, known for their antioxidant properties. Volatile phytochemicals anethole and \(\beta\)-caryophyllene that were found in all extracts have previously shown potential as antibacterial and antifungal agents. In the future, the extraction procedure could be optimised to fully utilise the Estonian _Galium vermum_ (especially the blossoms) as a rich source for asperuloside. Though the hydroacetonic extract showed higher yields of asperuloside in the current study, the hydroethanolic yields were not significantly lesser. For potential future purposes, this presents an important advantage, as ethanolic extracts are suitable for biological applications. In conclusion, _Galium_ species native to Estonia should be considered for future therapeutic approaches due to their many favourable properties. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/molecules28062867/s1](https://www.mdpi.com/article/10.3390/molecules28062867/s1)]([https://www.mdpi.com/article/10.3390/molecules28062867/s1](https://www.mdpi.com/article/10.3390/molecules28062867/s1)). The graph illustrating the correlation of polyphenic content and antioxidants of the extracts (Figure S1) as well as the UV spectra of all quantified phytochemicals measured from the analyses of 50% ethanol extracts (Figures S2-S29) are provided as a separate file. **Author Contributions:** Conceptualization, M.V. ; methodology, P.S.-R., P.J., M.V. ; formal analysis, P.-R.L., P.S.-R., P.J., O.B. ; investigation, P.-R.L., P.S.-R., M.V. ; data curation, P.-R.L., P.S.-R.; writing--original draft preparation, P.-R.L., P.J.