4 dimethylaminocinnamaldehyde dmaca

All chemicals were at least of analytical grade. Acetic acid, formic acid, hydrochloric acid (37%), toluene, 1-propanol, and 4-dimethylaminocinnamaldehyde (DMACA) were from Merck (Darmstadt, Germany). Acetone, ethanol as well as HPLC grade acetonitrile and methanol were purchased from Sigma-Aldrich (St. Louis, MO, USA). LC–MS grade acetonitrile and methanol used for MS analyses were from Fluka (Buchs, Switzerland). MilliQ 18.2 MΩ water (Millipore, Bedford, MA, USA) was also used.
Standard of (−)-epicatechin was obtained from Sigma-Aldrich and (+)-catechin from Carl Roth (Karlsruhe, Germany). Standards of procyanidin B1, procyanidin B2, procyanidin B3, procyanidin C1, (−)-catechin gallate, (−)-gallocatechin, (−)-gallocatechin gallate, (−)-epicatechin gallate, (−)-epigallocatechin and (−)-epigallocatechin gallate were obtained from Extrasynthèse (Genay, France).

The Azolla fronds were fixed in 3% (w/v) glutaraldehyde in 0.075-M phosphate buffer, pH 7.2, for 24 h. Each of the samples were then washed three times for 7 min in the 0.075-M phosphate buffer, pH 7.2, and post-fixed in 1% (w/v) OsO₄ for 1.5 h. The samples were then dehydrated in increasing ethanol concentrations and embedded in resin (Epon, 2-dodecenylsuccinic anhydride, and methylnadic anhydride mixture). Semi-thin sections (1–2 μm) were cut with an ultramicrotome (OmU2; Reichert, Heidelberg, Germany) equipped with a glass blade, stained with toluidine blue, and observed using a light microscope (BX53; Olympus, Tokyo, Japan) (Reale et al., 2017 (link)). Red Azolla fronds taken from the botanical garden were free-hand sectioned using a razor blade. They were then mounted on slide glasses with a drop of distilled water and examined in a bright field using the light microscope. The staining of flavan-3-ols and PAs with 4-dimethylaminocinnamaldehyde (DMACA) (Merck Life Science, Milan, Italy) was basically performed as reported in Abeynayake et al. (2011 (link)).

All solvents were at least of analytical grade. Methanol (HPLC and LC-MS grade), acetone, and acetonitrile (LC-MS grade) were obtained from Honeywell Reagents (Seelze, Germany). Ethanol (absolute anhydrous) was purchased from Carlo Erba Reagents (Val de Reuil, France). Ethyl acetate, acetic acid (glacial (100%) and glacial (100%) LC-MS grade), concentrated hydrochloric acid (37%), and 4-(dimethylamino)cinnamaldehyde (DMACA) were acquired from Merck (Darmstadt, Germany). Ammonium acetate, 2,2-diphenyl-1-picrylhydrazyl (DPPH), (−)-epicatechin (90%), and (−)-catechin (98%) were acquired from Sigma-Aldrich (Steinheim, Germany). Ascorbic acid was obtained from Fluka, Sigma-Aldrich (Steinheim, Germany). (−)-Epicatechin (of high purity) was purchased from Fluka Chemie (Buchs, Switzerland), while (+)-catechin (98%) was obtained from Carl Roth (Karlsruhe, Germany). A Milli-Q water purification system (18 MΩ cm⁻¹; Millipore, Bedford, MA, USA) was used to obtain ultrapure water. Disposable plastic cuvettes were purchased from Brand (Wertheim, Germany).