*2.2. Characterization of Polar Bioactive Compounds from C. hypocistis Extracts by UPLC-ESI-QTOF-MS*

Following the described LC-MS method, all extracts were analysed, resulting in a total of 148 detected compounds. Figure 2 shows the base peak chromatograms performed for each extraction condition and Table S2 summarises all information about detected compounds such as retention time, *m*/*z* ratio, error in ppm, molecular formula, and name of each proposed compound. In addition, peak numbers were assigned according to their elution order. The peak areas of the detected compounds are given in Table S3.

It is worth to note that to our knowledge, there is little reference to a comprehensive characterisation of *C. hypocistis* extracts [1]. For this reason, our work is especially relevant. Considering the accurate mass spectra information and data previously reported by literature, 136 compounds were tentatively annotated in this study. Only one common molecular feature was detected among all the different extractions; however, this molecular feature could not be annotated and remained as an unknown compound (Table 1). This could be explained mainly by the differences in the extraction efficiencies of the different solvents used, also considering the different physicochemical properties of the compounds present in the matrix.

**Figure 1.** Total phenolic and flavonoid content (**A**), radical scavenging ability (**B**), reducing power (**C**), metal-chelating ability (MCA) (**D**), total antioxidant ability (by phosphomolybdenum assay (PBD)) (**E**), and Pearson's correlations between total bioactive compounds and antioxidant assays (*p* < 0.05) (**F**). na: not active; different letters in column for same assays indicate significant differences in the extracts (*p* < 0.05).

Overall, the tentative characterisation allowed one to classify the compounds in five major groups: gallotannins, ellagitannins, flavonoids, fatty acids, and other compounds, with it being important to note that gallotannins were the most important one, with 61 compounds included. These were mainly annotated as mono-, di-, tri-, tetra-, penta-, hexa-, and hepta-galloyl hexoside. In addition, different isomers were also annotated for each of these types of chemical structures. Silva et al., reported in 2020 from mono- to penta-galloyl hexosides in different parts (petals, stalks, and nectar) of *C. hypocistis* [24]. However, all these compounds were previously reported in other sources such as *Magnifera indica* L. kernels and peels [25,26], *Rhodiola crenulate* roots [27], *Rhodiola rosea* roots, leaves, stems and flowers [27], Paeonia plants [28], and *Pistacia vera* leaves [29], among others [30]. Table 1 shows that a major number of these gallotannins were found in ethyl acetate, ethanol, water/ethanol, and water extracts. In recent years, several authors have reported a wide range of biological properties of these galloyl hexoside derivatives [27,29,31,32]. Among other gallic acid derivatives presented in the gallotannins group, three isomers from neochebulagic acid corresponding to peaks 60, 61, and 70 have been also annotated. These compounds were reported in *Terminalia chebula* Retz. Playing a role in the intestinal glucose transport [33]. Moreover, several compounds previously found in *Trapa quadrispinosa* pericarps were detected in our extracts, such as peaks 49, 67, 75, and 87 (digalloyl-lactonised valoneoyl-d-glucose isomers); peaks 71, 92, and 98 (trigalloyl-lactonised valoneoyl glucose isomers); and peaks 78, 85, and 102 (galloyl-penta-hydroxy-benzoic-brevifolincarboxylglucose isomers) [34].

**Figure 2.** Base peak chromatograms from Cytinus (**a**) hexane, (**b**) ethyl acetate, (**c**) dichloromethane, (**d**) ethanol, (**e**) ethanol/water, (**f**) water, (**g**) NADES-A, (**h**) NADES-B, and (**i**) NADES-C extracts by UPLC-MS.


**Table 1.** Chemical characterisation of the tested extracts.




*Molecules* **2022** , *27*, 5788





isomer 3





present; ND: nondetected.

As the second most important group, ellagitannins contains 30 compounds, which were annotated as different mono-, di-, and tri-galloyl-DHHDP-glucose isomers and digalloyl-HHDP-iso DHDG-glucose isomers. Besides these compounds, other ellagitannins such as terflavin B, phyllanthussin C, geraniin, and balanophlorotannin E isomers have been previously reported in various *Terminalia* species and in *Trapa* species [34,35]. In case of our species, only peaks corresponding to *m*/*z* 937 and 783 were annotated [24].

Regarding flavonoids, catechin and epicatechin as flavan-3-ols were found; quercetin and two isorhamnetin glucoside isomers as flavonols were also detected, mostly with water, ethanol, their mixture, and ethylacetate.

On the other hand, hexane and dichloromethane extracts presented the major number of fatty acids (Peaks 118, 119, 121–132, 135–141, 144–148). Regarding NADESs, the three extracts showed the lower number of features, and many of the features obtained could not be annotated such as peaks 7 and 9 (unknowns 3 and 4) were only found in NADES-C extract. The same happened for peak 2 (unknown 2); this compound was only detected in NADES-B. Among them, NADES-A presented high number of features corresponding mainly to gallotannins. This highlights the potential of NADESs to obtain extracts containing additional bioactive compounds and to "tailor" the properties of extracts. Secondly, extraction by two NADESs sequentially should allow for the fractionation of bioactive compounds.

Other compounds have been also annotated, although they have not been classified in specific groups due to the low number and the high range of structures. For instance, organic acids (quinic and fukiic acids); simple phenols (gallic and ellagic acids); and two alkyl-phenylketones, namely brevifolin and brevifolin carboxylic acid [36], were included in this group. In addition, three isomers from galloylnorbergenin, antioxidant isocoumarins that were previously found in leaves of *Diospyros gilletii* De Wild [37] were also included.
