Variability in the composition of phenolic compounds in winter-dormant Salix pyrolifolia in relation to plant part and age Author Lavola, Anu ∗ & Department of Environmental and Biological Sciences, Natural Product Research Laboratories, University of Eastern Finland (UEF), P. O. Box 111 FI- 80101 Joensuu, Finland Author Maukonen, Merja Author Julkunen-Tiitto, Riitta text Phytochemistry 2018 2018-09-30 153 102 110 http://dx.doi.org/10.1016/j.phytochem.2018.05.021 journal article 10.1016/j.phytochem.2018.05.021 1873-3700 10483857 2.1. Phytochemicals in winter-dormant S. pyrolifolia In this study, the main phenolic compounds of clonal S. pyrolifolia tree were analysed from the different plant parts of winter-dormant twigs. The phenolic profile of the twigs was mainly composed of the salicylate glucosides and other Salix -specific, simple phenolic glucosides but also, several types of flavonoid glycosides and phenolic acids were detected ( Table 1 , Fig. 1 ). These low molecular-weight (LMW) compounds comprised about 20% of the dry weight in twigs ( Fig. 2 ). In addition, all the plant parts contained high molecular-weight condensed tannins (proanthocyanidins), which comprised about 10% of the dry wt in twigs ( Fig. 2 ). This high content of phytochemicals may be suggested to be partly attributable to the high resistance that S. pyrolifolia shows to microbes and herbivores (e.g. Boeckler et al., 2011; Riitta Julkunen-Tiitto, pers. observation). Salicylates (salicinoids), the derivatives of salicyl alcohol with β- Dglucopyranose moieties, as well as some other simple glucosides derived from cinnamic acid, are typical compounds for Salix species. In here, the winter-dormant twigs of S. pyrolifolia were composed of four different types of salicylate glucosides: salicin, HCH-derivative (hydroxycyclohexenone) of salicin, salicortin and disalicortin ( Table 1 ). Salicin (glucoside of salicyl alcohol) is the most widespread and simplest glucoside in Salix species , which is detected in fairly low amounts in leaf tissues but may occur in quantities in bark (Heiska et al., 2007; Julkunen-Tiitto, 1989a , 1986 ; Pob ł ocka-Olech et al., 2007), in buds ( Julkunen-Tiitto, 1989a ; Sivadasan et al., 2015 ) and also, is found in seeds ( Randriamanana et al., 2015b ). Additionally, most Salix species contain high amounts of salicortin, the HCH-ester of salicin, while only a few species are rich in its derivatives, acetylsalicortin or tremulacin (e.g. Boeckler et al., 2011; Meier et al., 1988 ; Julkunen-Tiitto, 1989a ). In winter-dormant S. pyrolifolia , salicortin is the most abundant individual compound, and especially in bark, its concentration is extremely high, comprising over 10% of the dry wt ( Table 2 ). Another HCH-derivative of salicin with the UV-spectrum similar to salicortin but much longer retention time, and which molecular structure is suggested to consist of salicin with the HCH moiety attached to glucose ( Fig. 1 ) due to the accurate molecular mass of salicin + HCH based on the QTOF-analysis, is preliminary characterised here for the first time from the twigs of Salix species ( Table 1 ). The non-salicylate-based, simple phenolic glucosides in S. pyrolifolia were salireposide, triandrin (the glucoside of coumaroyl alcohol), picein ( p -hydroxyacetophenone glucoside) and its derivative. These compounds are generally regarded as the twig-specific components of Salix species (e.g. Julkunen-Tiitto, 1989a ; Meier et al., 1988 ). The concentrations of both picein and triandrin fluctuated among the sampled twigs and in some samples, triandrin was found only in trace quantities ( Tables 2 and 3 ). The high within-species and intra-plant variation in the concentrations of these simple phenolic glucosides is a commonly reported phenomenon in Salix species (e.g. Heiska et al., 2007; Ikonen, 2002; Julkunen-Tiitto, 1989b ; Nybakken and Julkunen-Tiitto, 2013 ; Nyman and Julkunen-Tiitto, 2005 ; Sulima et al., 2017 ). For the most part, the composition of both salicylate glucosides and simple phenolic glucosides in this study was in accordance with previous analyses from S. pyrolifolia ( Julkunen-Tiitto, 1989a ,b). Here, however, we did not find acetylsalicin (fragilin) or acetylsalicortin in the samples, but on the other hand, we identified the HCH-derivative of salicin, disalicortin, triandrin and salireposide as new components in S. pyrolifolia twigs. Salireposide has so far been detected in only a few Salix species : S. myrsinifolia , S. petiolaris , S. purpurea and S. rosmarinifolia (e.g. Boeckler et al., 2011; Julkunen-Tiitto, 1989a ; Meier et al., 1988 ; Nybakken and Julkunen-Tiitto, 2013 ). The flavonoid composition among Salix species is found to be varied and anthocyanins, flavonols, flavones, flavanones and chalcones are the different groups of flavonoids determined in the genus Salix (e.g. Bridle et al., 1970; Jarrett and Williams, 1967 ; Julkunen-Tiitto and Sorsa, 2001 ; Krauze-Baranowska et al., 2013 ; Nyman and Julkunen-Tiitto, 2000 , 2005 ). In winter-dormant S. pyrolifolia twigs, the flavonoids were mainly composed of different derivatives of naringenin (flavanones) and isosalipurposide (phloridzin, chalcone 2′- O -glucoside), but also, the derivatives of quercetin and kaempferol (flavonols), the derivatives of luteolin (flavone), a dihydroflavonol ampelopsin, and a flavan-3-ol catechin were identified ( Table 1 ). Two new isosalipurposide methoxyderivatives (monomethyl- and dimethylisosalipurposide) were also identified in S. pyrolifolia . Generally, the concentration of flavonoids in S. pyrolifolia was relatively high ( Fig. 2 , Table 2 ), and similar levels, over 1% of the dry wt in bark, have previously been reported only in S. acutifolia , S. daphnoides , S. purpurea and S. hastata ( Krauze-Baranowska et al., 2013 ; Meier et al., 1992 ). In these species, the main flavonoid glucosides in twigs were also the same as in S. pyrolifilia : naringenin 7- O -glucoside (prunin), naringenin 5- O -glucoside (salipurposide), a derivative of naringenin 5- O -glucoside and isosalipurposide ( Jarrett and Williams, 1967 ; Kammerer et al., 2005 ; Krauze-Baranowska et al., 2013 ; Meier, 1988; Pob ł ocka-Olech et al., 2007; Sulima et al., 2017 ). Ampelopsin (dihydromyricetin) has previously been reported to be the major leaf component in S. phylicifolia , and it is also found from the leaves of S. lapponum ( Nyman and Julkunen-Tiitto, 2000 ; Tegelberg et al., 2003 ), in the bark of S. daphnoides , S. purpurea and S. pentandra (Förster et al., 2008) , and in the wood of S. caprea ( Pohjamo et al., 2003 ) . The phenolic acid composition in winter-dormant S. pyrolifolia twigs consisted mainly the derivatives of p -hydroxycinnamic acids ( p -coumaric acids) and chlorogenic acids (caffeoylquinic acids, CQA), and several of them were not able to get identified in our analytical conditions ( Table 1 ). Similarly, the bark of S. myrsinifolia and S. purpurea has reported to contain different kinds of p -hydroxycinnamic and chlorogenic acid derivatives but also, the derivatives of benzoic acids have been found from the bark of S. purpurea ( Nybakken and Julkunen-Tiitto, 2013 ; Pob ł ocka-Olech et al., 2010). The phenolic acid composition in Salix species has turned out to be diverse and very plant part-specific (e.g. Nissinen et al., 2016 ; Sivadasan et al., 2015 ; Tegelberg et al., 2003 ).