Persicaria ((L.)) Mill.

Prota, N., Mumm, R., Bouwmeester, H. J. & Jongsma, M. A., 2014, Comparison of the chemical composition of three species of smartweed (genus Persicaria) with a focus on drimane sesquiterpenoids, Phytochemistry 108, pp. 129-136 : 130

2.1. Interspecific variation of the chemical composition of Persicaria spp.

Seeds of three species of the Persicaria genus, P. hydropiper , P. minor and P. maculosa , were collected in the woods in close range of each other. The three species were identified by taxonomist Ronald van den Berg (Wageningen UR) (Supplementary Fig. S3 for images of the plants). Seven separate samples were taken from plants raised from the seeds: three developmental stages of flowers and four of leaves. In Fig. 1 , a cross section of a P. hydropiper flower clearly shows the presence of the valvate glands in the tepals of the flowers. Samples were extracted in dichloromethane (DCM) and analysed by coupled gas chromatography–mass spectrometry (GC–MS). A total of 29 major metabolites could be identified across all three species, 15 of which were present in all. Nearly all (27/29) identified metabolites were terpenoids, most were sesquiterpenoids (21/29) and a quarter of those were drimanes (5/21) ( Tables 1 and 2 , structures in Fig 2 , and mass spectra in Fig. S1 ). Table 1 shows the average relative abundance of the 29 metabolites in the flowers of the three species, while in Table 2 the chemical profiles of the leaves are listed. P. hydropiper was the species with the widest variety of metabolites detected in our analyses; the only compound present in the other two species, but not in water-pepper, was b- selinene. In general, flower samples contained more different and higher quantities of metabolites compared to leaves, with the exception of the three neophytadiene isomers, which were more abundant in the leaves, because they are degradation products of chlorophyll ( Rowland, 1957).

The five sesquiterpene drimanes observed in the gas chromatogram were drimenol, polygodial, 9- epi -polygodial, drimenin and isodrimenin ( Fig 2 ). It is known that 9- epi -polygodial is formed from polygodial by base-treatment ( Cortés et al., 1998; Kubo and Ganjian, 1981), and there are other reports of this compound being an artifact of the GC–MS analysis ( Asakawa et al., 2001). We confirmed that upon injection of a 1 H NMR verified reference standard of pure polygodial, also 9- epi -polygodial is observed, and that the ratio of the peaks versus 9- epi -polygodial varied from 1.6 to 0.8:1 depending on the injection temperature ( Fig. S2 ). An earlier study, however, reports the 1 H NMR spectra for both epimers in Drymis winterii ( Rodríguez et al., 2005) . As our GC–MS method does not allow the reliable estimation of the ratios, we report the combined concentrations of both epimers under the label of polygodial.

Polygodial was the most abundant compound in P. hydropiper flowers, making up for 77% of the extract, equivalent to 6.2 mg g FW — 1 ( Tables 1 and 3 ). It was 200 times less abundant in P. minor flowers (0.032 mg g FW — 1), representing 25% of the total compounds, and 100,000 times less abundant in P. maculosa flowers (70 ng g FW — 1), representing 0.1% of total compounds ( Tables 1 and 3 ). The drimane sesquiterpene lactones drimenin and isodrimenin were minor compounds when compared to polygodial, accounting for only 2% of the P. hydropiper flower extract. Relative to P. hydropiper , they were 10- and 100-fold less abundant in P. minor flowers, respectively, and not detected in P. maculosa flowers. Drimenol, the putative precursor of polygodial ( Pickett, 1985) was only detected at low concentrations in P. hydropiper and not in the other species, presumably because it serves as an intermediate and it is promptly converted. Comparing leaves to flowers, polygodial was 10 × more abundant in flowers of P. hydropiper , equally abundant in P. minor leaves and flowers and 10 × less abundant in flowers than leaves of P. maculosa .

The low levels of polygodial in P. maculosa were earlier not found by Hagendoorn et al. (1994) for specimens collected in the Netherlands, but they are in line with the results of Derita et al. (2008) who detected low amounts of polygodial (0.54 mg g DW — 1) in leaves of P. maculosa from Argentina. Yet, by comparison, our specimen contains only 70 ng g FW — 1. Taking into account the FW/DW comparison, this is still 100 times less than accessions from Argentina.

Figures

Fig. 3. Similarity matrix of the compounds, obtained using the Log2-transformed peak areas of the different P. hydropiper flower samples throughout development. The similarity of the abundance pattern between two given compounds assumes values between —1 and 1, with 1 meaning a 100% identical pattern. The cells at the diagonal are always intensely red as they represent the comparison of the abundance pattern of one compound with itself.

Fig. 1. Light micrograph of a longitudinal cross section of a paraffin embedded flower of P. hydropiper. Arrows indicate the valvate glands (cavities) where polygodial is stored.

Fig. 2. Structures of the five drimane sesquiterpenoids observed in the extracts of the analysed plants: (1) drimenol; (2) polygodial; (3) 9-epi-polygodial; (4) drimenin; (5) isodrimenin.