# Phytochemical Screening and Antioxidant Activity of Selected Estonian _Galium_ Species
## 2 Results and Discussion
### Identification and Quantification of Major Constituents in Extracts
Even though this could be stated to be true for Estonian species, the abundance of diosmetin isomers in our extracts distinctly differentiates the two chromatographic profiles as the corresponding peaks are much lesser in size for _Galium verum_ (Figure 1). [PERSON] et al. also analysed the iridoid patterns of the Bulgarian _Galium_ species and found that _Galium verum_, _aparine_ and _mollugo_ all contain asperulosidic acid [12], and this was confirmed by our data to also be true for the Estonian species (comp. 7). When studying the polyphenolic content of 70% ethanol extracts of Romanian _Galium_ species, [PERSON] et al. found that _Galium verum_, _aparine_ and _mollugo_ all contained chlorogenic acid, rutin, and quercetin [13]. Our research demonstrates that 50% acetone, 50% ethanol, and 80% ethanol extracts of these species all contain these polyphenols also (comp. 9, 14, 26). It is important to note that the abundance of these polyphenics between the different species is varied, and, in general, the use of a less polar solvent (50% acetone) helps enhance the quantities of polyphenics in the extracts (Table 2). Interestingly, kaempfer-7-O-glucose (comp. 16) was identified in both _Galium verum_ and _Galium mollugo_, whereas in the study of Bulgarian _Galium_ species by [PERSON] et al., only _Galium verum_ was shown to contain kaempfer glycosides [12]. This finding demonstrates how the phytochemical makeup of a species can vary based on the geographic origin. Our research also demonstrates the differences in phytochemical content dependant on the extraction solvent used (and its polarity), as 50% acetone, 50% ethanol, and 80% ethanol extracts showed varied yields of phytochemicals quantified (Table 3). The extracts of _Galium verum_ blossoms contained the highest amounts of the same two compounds, asperuloside and rutin, regardless of the extraction solvent used. Thirdly, either dicaffeoylquinic acid isomer (comp. 22; in the 50% acetone and 80% ethanol extract), or chlorogenic acid (in the 50% ethanol extract), was found. In the _Galium verum_ herb extract, the phytochemical in the highest abundance was also asperuloside, followed by chlorogenic acid, and thirdly, either deacetylasperulosidic acid (50% ethanol extract) or asperulosidic acid (50% acetone and 80% ethanol extracts). In _Galium aparine_ extracts, the most abundant phytochemical was either asperulosidic acid (50% acetone and 50% ethanol extracts) or asperuloside (80% ethanol extract). The second and third compound were similar in hydroethanolic extracts, chlorogenic acid and rutin, and differed in the hydroacetonic extract, rutin and asperuloside. Finally, in the _Galium mollugo_ extracts, the three most common phytochemicals were similar, but found in differing amounts for each extraction solvent: these compounds were chlorogenic acid (in highest abundance in 50% acetone); asperuloside (in highest abundance in 80% ethanol); and diosmetin isomer, comp. 19 (in highest abundance in 50% ethanol). In conclusion, 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. Asperuloside has shown to have a wide array of therapeutically beneficial qualities [51]. Hence, it is important to note that the species with the highest abundance of asperuloside is _Galium verum_, with the extracts of _Galium verum_ blossoms showing higher amounts of asperuloside compared to the extracts of the whole aerial part of the plant. Additionally, a less polar solvent should be preferred when extracting asperuloside from _Galium_ species, as the yields were shown to be more favourable with hydroacetonic extracts compared to the more polar, hydroethanolic extracts. On the other hand, it is noteworthy that the yields were not significantly less in the hydroethanolic extracts. This is an advantage for future biological applications, as acetonic extracts would not be suitable for such purposes.