Gallic acid derivatives, ellagitannins, quinic acid derivatives, kaempferol derivatives, quercetin derivatives, myricetin derivatives, procyanidins, and prodelphinidins were quantified from all the 18 cell cultures using the UHPLC–MS2 methods described earlier by Engström et al. (2014, 2015). The concentrations of each of the quantified polyphenol groups are presented in Table 2. All plant cell cultures except for R. idaeus contained at least trace amounts of procyanidins and gallic acid derivatives, the latter of which consisted mainly of monogalloyl glucose isomers in all samples. Suspension cultures from three species, S. aucuparia, V. myrtillus, and E. nigrum, proved exceptionally interesting in their polyphenol content both qualitatively and quantitatively. One culture from all three of these species was chosen for more accurate qualitative analyses by Orbitrap mass spectrometry.
A total of 10 cell suspension cultures were included from the family Rosaceae, representing three genera (Rubus, Fragaria, and Sorbus). From the genus Rubus, a total of seven cultures from four species (R. chamaemorus L., R. idaeus L., R. arcticus L., and R. saxatilis L.) were included. Rubus plants are most often characterized by high ellagitannin concentration in both their berries (Kähkönen et al. 2001; Nohynek et al. 2006) and leaves (Okuda et al. 1992), with the main ellagitannins typically being the dimeric sanguiin H-6 and the trimeric lambertianin C. Other phenolic compounds in R. idaeus include anthocyanins in fruits and flavonols, flavan-3-ols, proanthocyanidins, ellagic acid conjugates, and phenolic acids in both fruits and leaves (Harborne and Hall 1964; Ryan and Coffin 1971; Henning 1981; Törrönen et al. 1997; Kähkönen et al. 2001; Määttä-Riihinen et al. 2004b). Both fruits and leaves of the other three Rubus species included in the study are known to include very similar compounds to R. idaeus (e.g., Okuda et al. 1992; Törrönen et al. 1997; Häkkinen et al. 1999; Kähkönen et al. 2001; Määttä-Riihinen et al. 2004b).
In contrast, from the cultures included in this study, only the ones originating from R. saxatilis contained detectable levels of ellagitannins. In addition, the ellagitannins observed were determined to be monomers, and no traces of the characteristic oligomeric ellagitannins were detected. Besides ellagitannins, gallic acid derivatives were detected in all Rubus cultures except R. idaeus. Small amounts of proanthocyanidins were detected in five out of seven of the Rubus cultures, and flavonoids only in R. arcticus cultures. Anthocyanins were detected in Ra1 as cyanidin, delphinidin, petunidin, and peonidin glucosides, galactosides, and/or arabinosides.
Fragaria × ananassa (Duchesne ex Weston) Duschesne ex Rozier ‘Senga Sengana’ was represented by one culture, and it proved to be fairly similar to several of the Rubus cultures in that it mainly consisted of small amounts of procyanidins and trace quantities of gallic acid derivatives. Earlier, López Arnaldos et al. (2001) have studied the changes in total soluble phenolics and flavanols, (+)-catechin, and ferulic acid and its glucoside in F. × ananassa callus cultures during their growth. They found that the concentrations of phenolic compounds peaked in the beginning of the exponential growth phase. As can be noted, the compounds they detected were not in line with the compound groups we identified to be present in our F. × ananassa suspension culture, which is most likely explained by different culture conditions, age, heritage of the culture, and different cultivar.
The phenolic content of the fruits of F. × ananassa has been studied fairly thoroughly, and they are known to contain ellagitannins and ellagic acid derivatives, proanthocyanidins, anthocyanins and other flavonoids including flavonols and flavan-3-ols, and hydroxycinnamic acids (Gil et al. 1997; Häkkinen et al. 1999; Aaby et al. 2007; Buendía et al. 2010).
The leaves of F. × ananassa, on the other hand, are not nearly as studied as the fruits, but they are known to contain ellagitannins, ellagic acid derivatives, galloyl glucoses, proanthocyanidins, flavonoids, and hydroxycinnamic acids (Skupień and Oszmiański 2004; Kårlund et al. 2014).
Two S. aucuparia L. suspension cultures cultivated by different methods were included. The culture Sa1 was cultivated in shake flasks, whereas the culture Sa2 was grown in a plastic cultivation bag in a wave type bioreactor. Their phenolic profiles were similar, consisting mostly of galloyl glucoses, ellagitannins, and procyanidins. Quantitatively culture Sa2, which was grown in a wave type bioreactor, had higher concentrations of both gallic acid derivatives and ellagitannins, showing its higher potential to produce these successive compound groups of the hydrolysable tannin pathway. The culture grown in shake flasks, Sa1, produced ellagitannins in trace quantities only, as did both S. aucuparia cultures procyanidins. The UV chromatogram (λ = 280 nm) of the S. aucuparia culture Sa2 is presented in Fig. 1 and a more detailed characterization of the compounds detected in it is presented in Table 3. The main ellagitannins were identified as two isomeric galloyl-bis-HHDP-β-d-glucopyranoses and one trigalloyl-HHDP-β-d-glucopyranose. Galloyl glucoses from monogalloyl glucose to di-, tri-, tetra-, and pentagalloyl glucoses were detected as well (Fig. 2), with mono- to trigalloyl glucoses appearing as several isomers.
The identities of these hydrolysable tannins were further confirmed by MS2 experiments. Galloyl glucoses showed characteristic fragments at m/z values 169 and 125, corresponding to gallic acid and a subsequent cleavage of its CO2 unit (Lee et al. 2005). Fragments resulting from the cleavage of a galloyl or a gallic acid unit were detected, resulting in a loss of 152 or 170 Da from the precursor ion, respectively. Ellagitannins were similarly detected by the presence of ions at m/z values 301, 275, and 249 corresponding to ellagic acid, and a further loss of either one or two CO2 units, and a corresponding loss of 302 Da from the precursor ion. The fragments presented for galloyl glucoses are often observed with ellagitannins, as they can also include galloyl groups. The identity of peak 2 could not be solved, but the accurate mass measurements suggest that it might contain two nitrogen atoms, making it not a true polyphenol, and that the two detected ions are [M−H]− and [2M−H]−. Peaks 4 and 6 are very likely related to each other, as they have similar fragmentation patterns, and they might be hexosides, as the ion at m/z 223, resulting from a 162 Da loss from the likely [M−H]− ion at m/z 385, was of high intensity.
The compounds identified are in clear contrast to S. aucuparia berries, the phenolic profile of which has been reported to consist of hydroxycinnamic acids, hydroxybenzoic acids, anthocyanins, procyanidins, flavonols, and flavanols (Kähkönen et al. 2001), with 3-caffeoylquinic acid being the main compound contributing to 46% of 18.83 mg g−1 DW of total phenolics (Kylli et al. 2010). The leaves and inflorescences of S. aucuparia have not been studied as widely as the berries, but they contain phenolic acids, proanthocyanidins, and flavonoids (Olszewska and Michel 2009).
The profiles of the studied Rosaceae species cultures shared some similarities. Their average gallic acid derivative concentrations were higher than in the cultures of other plant families, and ellagitannins were only detected in Rosaceae cultures, albeit only in two of the six included species. This is in line with the fact that plants in the family Rosaceae are generally rich sources of hydrolysable tannins (Moilanen et al. 2015), and the possibility of using oligomeric ellagitannins as chemotaxonomic markers in Rosaceae has been suggested by Okuda et al. (1992), for example. All of the galloyl glucose producing Rosaceae cell cultures contained 1-O-monogalloyl-β-d-glucose, which is biosynthetically the first hydrolysable tannin, a precursor of all other hydrolysable tannins, and, therefore, an important intermediate in the hydrolysable tannin pathway. It remains unknown why it was not efficiently converted to biosynthetically following hydrolysable tannins.
A total of four cell suspension cultures were included from the family Ericaceae, encompassing two genera (Vaccinium and Empetrum) and three species. V. myrtillus L. proved to be the most efficient producer of procyanidins among all studied suspension cultures by far; two V. myrtillus cultures were studied, and they contained 20.88 mg g−1 DW and 26.26 mg g−1 DW of procyanidins, contributing to 98% of total polyphenols. Several A-type proanthocyanidins were detected, which is in line with what has been found in V. myrtillus in nature (Hokkanen et al. 2009). As for other proanthocyanidins, prodelphinidins were found in much smaller quantities with 0.17 mg g−1 DW and 0.18 mg g−1 DW. Other compounds found in the V. myrtillus cultures were quercetin and kaempferol derivatives and gallic acid derivatives. The UV chromatogram (λ = 280 nm) of the V. myrtillus culture Vm2 is presented in Fig. 3 alongside with a more detailed characterization of the compounds in Table 4.
The characteristic ions for the detected proanthocyanidins result from several types of cleavages; the ion at m/z 407 results from retro-Diels–Alder fragmentation and subsequent elimination of water (Friedrich et al. 2000), ions at m/z 289 and 287 result from quinone methide cleavage (Friedrich et al. 2000; Karonen et al. 2011), ion at m/z 245 likely from the loss of –CH2–CHOH group from a catechin unit (Pérez-Magariño et al. 1999), and ion at m/z 125 corresponds to phloroglucinol resulting from heterocyclic ring fission (Gu et al. 2003). The identities of peaks 4 and 1 are tentatively identified as coumaroyl hexose and a coumaroyl hexose derivative, respectively, due to their mass fragmentation patterns showing signals at m/z values 163 and 119 possibly resulting from coumaric acid and further cleavage of CO2 (Ma et al. 2007). Peak 12 was identified as a quercetin hexoside based on its UV spectrum and a product ion at m/z 300, resulting from the homolytic cleavage of the O-glycosidic bond of the hexose (Hvattum and Ekeberg 2003). In addition, a product ion at m/z 301 corresponding to the quercetin aglycone was detected in the UHPLC-DAD–3Q-MS2 analyses, confirming its identity.
Beside the sharp peaks listed in Table 4, a chromatographic hump is visible in the chromatogram in Fig. 3 between approximately 3 and 6 min. This corresponds to a mixture of a multitude of different isomers of proanthocyanidin oligo- and polymers, which are not resolved when using reversed-phase liquid chromatography, except for some small oligomers. The mean degree of polymerization for the proanthocyanidins in this hump, however, can be determined using MS2 methods for both procyanidins and prodelphinidins (Engström et al. 2014). For both of the two V. myrtillus culture samples, these mean degrees of polymerization were calculated to be 5.
The berries of V. myrtillus have long been known to be rich in anthocyanins (Suomalainen and Keränen 1961). They include five different anthocyanidin aglycones (cyanidin, delphinidin, peonidin, petunidin, and malvidin), all appearing with three glycones (arabinose, glucose, and galactose), giving it a characteristic fingerprint profile of 15 different anthocyanins, which has for example been suggested for use in V. myrtillus product authenticity studies (Primetta et al. 2013). The two studied V. myrtillus cell cultures differed from this profile slightly, as no malvidin aglycones were detected. All of the other four aglycones and their corresponding three types of glycosides were detected.
Besides anthocyanins, the phenolic compounds of V. myrtillus berries include other flavonoids, proanthocyanidins, hydroxycinnamic acids, and ellagic acid (Wildanger and Herrmann 1973; Törrönen et al. 1997; Häkkinen et al. 1999;).
The phenolic compounds of the leaves, similarly to berries, of V. myrtillus include anthocyanins, proanthocyanidins, flavonoids, hydroxycinnamic acids, coumaroyl iridoids, and cinchonains (Riihinen et al. 2008; Hokkanen et al. 2009). The leaf anthocyanins are present only in the red leaves of V. myrtillus, which result from exposure to sunlight and subsequent accumulation of anthocyanins to protect from UV-B radiation (Chalker-Scott 1999; Jaakola et al. 2004).
The polyphenol profile of the V. vitis-idaea L. culture was somewhat similar to that of the V. myrtillus culture, containing mostly procyanidins and quercetins, though especially procyanidins in much lower concentrations. The phenolic profile of the leaves of V. vitis-idaea has been determined to mostly consist of flavonoids, including catechin and epicatechin, and simple phenolic acids, with some proanthocyanidins, cinchonains, and coumaroyl iridoids (Ek et al. 2006; Hokkanen et al. 2009). The berries contain all of these, with the addition of anthocyanins (Andersen 1985; Häkkinen and Auriola 1998; Määttä-Riihinen et al. 2004a; Ek et al. 2006).
The E. nigrum L. culture contained the highest concentration of prodelphinidins of all samples, with 2.03 mg g−1 DW. Its procyanidin content was higher than that (3.04 mg g−1 DW), but still distinctly less than the procyanidin content in V. myrtillus. Similar to V. myrtillus, the mean degree of polymerization of proanthocyanidins was calculated, and determined to be 2, showing clearly smaller oligomers compared to V. myrtillus samples on average. Anthocyanins with four different aglycones (cyanidin, delphinidin, peonidin, and petunidin) were detected as well. The UV chromatogram (λ = 280 nm) of the E. nigrum culture En is presented in Fig. 4 alongside with a more detailed characterization of the compounds in Table 5. Fragmentation patterns of the E. nigrum sample proanthocyanidins are very similar to the ones observed and described for V. myrtillus, and some ions are seen with 16 Da larger m/z values due to the additional hydroxylation of the B-ring in prodelphinidins compared to procyanidins. The coumaric acid derivatives and the quercetin hexose were similar as in V. myrtillus, and the caffeoyl hexoses (Roche et al. 2005) and naringenin hexoside (Sánchez-Rabaneda et al. 2004) were identified using the MS2 fragmentation data. Peak 14 remained unidentified.
The lipophilic phenolic extracts of E. nigrum leaves have been detected to contain chalcones, dihydrochalcones, and dihydrophenanthrene derivatives (Wollenweber et al. 1992), with the hydrophilic phenolic compounds of berries including hydroxycinnamic acids, flavonoids, proanthocyanidins, and high amounts of a wide range of anthocyanins (Määttä-Riihinen et al. 2004a).
The only suspension cultures that could produce proanthocyanidins in amounts comparable to that detected in natural plants were all from the family Ericaceae. In addition, all Ericaceae cultures were able to produce at least some amounts of flavonoids similar to their natural counterparts, as evidenced by the presence of kaempferol and quercetin derivatives.
As for the ratio of procyanidins to prodelphinidins, the detected 40:60 ratio of procyanidins to prodelphinidins in the E. nigrum culture is close to the ratio reported in E. nigrum berries (Määttä-Riihinen et al. 2004a). Furthermore, it is clearly different from the ratios in genus Vaccinium cultures, the proanthocyanidins of which consist almost exclusively of procyanidins.
Families Caprifoliaceae and Poaceae
The L. caerulea L. var. kamtschatica (family Caprifoliaceae) culture was exceptional in that it contained by far the largest concentration of quinic acid derivatives in all of the samples, with only one other sample containing any at all. Quinic acid derivatives also contributed to the majority of its polyphenol profile, with trace amounts of gallic acid derivatives and procyanidins detected.
The taxonomy of some Lonicera species and the varieties of L. caerulea is not completely settled, and therefore, also the literature on L. caerulea var. kamtschatica is at times ambiguous. The berries contain high quantities of anthocyanins, along with other flavonoids, proanthocyanidins, and phenolic acids (Terahara et al. 1993; Chaovanalikit et al. 2004; Jurikova et al. 2011), while the leaves contain flavonoids and phenolic acids (Oszmianski et al. 2011).
Both the two included cereal cultures from family Poaceae, A. sativa L. and H. vulgare L. were low in the analyzed polyphenols, with trace amounts of gallic acid derivatives and procyanidins in both and trace amounts of quinic acid derivatives in one of the H. vulgare cultures. The phenolic content of A. sativa has been reported to consist mostly of other types of phenolics, mainly avenanthramides (Collins 1989), which are phenolic alkaloids, and simple phenolic acids (Durkee and Thivierge 1977). H. vulgare has also been studied for its phenolic content, and proanthocyanidins and flavonoids contribute to the majority of the phenolic compounds not bound to the cell wall (McMurrough et al. 1996; Ferreres et al. 2009).
As described, the suspension cultures used in this study were mostly grown under similar conditions, with the exception of the Poaceae samples As and Hv1. The growth media, however, differed between the samples (Table 1), with a total of six different media being used, which was due to the fact that the conditions were not optimized for polyphenol accumulation, but for general growth. The cultivation of the plant cells also differed for some samples as has been described earlier; most were cultivated in Erlenmeyer flasks, but different cultivation methods were used for Rc1, Rc2, Sa1, and Sa2. The accumulation of phenolic compounds in cell cultures is highly dependent on the concentrations of the plant growth regulators, auxins, and cytokinins, and their ratio (Dias et al. 2016). As for polyphenolic compounds, other published examples of the effects of these types of adjustments to the conditions include the inhibition of the production of ellagitannins by NH4
+ in the medium (Ishimaru and Shimomura 1991) and the accumulation of anthocyanins and proanthocyanidins when using appropriate concentrations of sucrose (Decendit and Mérillon 1996). It must be noted that all these choices along with, for instance, the age of the culture and sub-culturing can cause the polyphenol profile to be vastly different even within a species. All of these naturally also influence how closely the cell cultures resemble their wild counterparts. Depending on these factors, reports on the polyphenolic profile of cell cultures may closely match that of the wild plants’ certain plant parts (e.g., our samples Vm1 and Vm2, Decendit and Mérillon 1996), be somewhat similar (e.g., our samples Rs1 and Rs2, López Arnaldos et al. 2001), or even remarkably different (e.g., our samples Sa1 and Sa2, Nohynek et al. 2014), making comparisons between different studies sometimes troublesome. Therefore, our results are not fully comparable in all cases to each other or to other culture studies involving the same species.