Deuterium oxide

A 540 µL aliquot of each saliva sample was mixed with 60 µL of NMR buffer in 5 mm outside diameter (OD) NMR tubes. NMR buffer for saliva was prepared as follow: 3 parts (V/V) deuterium oxide (99.9 atom % D, to provide a field frequency lock) (Sigma-Aldrich Corp. Milwaukee, WI, USA), and 1 part (V/V) stock of 2 mM of trimethylsilylpropanoic acid (TSP). The pH was adjusted to 7.4. Due to the low flow of plasma by finger prick collection, 90 µL of each plasma sample was mixed with 90 µL of NMR buffer in 3 mm OD NMR tubes. NMR buffer for plasma was prepared as follows: 1 part (V/V) deuterium oxide (99.9 atom % D, to provide a field frequency lock) (Sigma-Aldrich Corp. Milwaukee, WI, USA), 2 parts (V/V) of ultrapure water, and 1 part (V/V) stock of 2 mM of TSP. The pH was also adjusted to 7.4. In addition to individual analysis of the samples, a pooled saliva sample (15 µL of each saliva sample) and pooled plasma sample (4 µL of each plasma sample) were analyzed to ensure unambiguous assignment and as an external standard to verify the stability of the automation in the NMR analysis.

ChemicalsExtract from Glycyrhizza glabra roots from FLOS (Mokrsko, Poland) was obtained by methanol extraction in a Soxhlet apparatus. Then, the extract was subjected to two nanofiltration processes, first using a 3-kDa (Merckmilipore, Darmstadt, Germany) membrane, then, the second, with the use of a 0.5-kDa membrane (TriSep, Sterlitech, Auburn, WA, USA). The process was conducted using Amicon Stirred Cell with a total volume of 200 mL (Merkmilipore). As a result, two fractions of extracts were used during the research, crude extract marked as GgC and purified extract (with molecules size between 3 kDa and 0.5 kDa), GgP.
In the presented studies, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE, 99%; from Avanti Polar Lipids (Alabaster, AL, USA)) was used as the film and liposome forming substance. Chloroform of high-purity Uvasol (Merck, Warszawa, Poland) was used to prepare the Langmuir monolayer. Dulbecco′s Phosphate Buffered Saline (Sigma Aldrich, Poznań wielkopolskie, Poland) was applied as a solvent to prepare saponins solution or as a subphase.
Deuterium oxide was purchased from Merck KGaA (Darmstadt, Germany).

Potassium phosphate monobasic (KH₂PO₄), sodium azide (NaN₃), and potassium hydroxide (KOH), were purchased from Sigma-Aldrich (St. Louis, Missouri, United States). Deuterium oxide and trimethylsilyl-2,2,3,3-tetradeuteropropionic acid (TSP) were acquired from Merck (Darmstadt, Germany).

Alkynes were purchased from Merck (Sigma‐Aldrich Company Ltd), The Old Brickyard, New Road, Gillingham, Dorset, SP8 4XT, UK, or were prepared by acylation of the appropriate alkynol with the corresponding acyl or sulphonyl chloride, using triethylamine as base, in dichloromethane. Deuterium oxide (99.9 atom%²H) and N,N‐dimethylformamide, DMF, (<0.005% water content) were also obtained from Merck (address above). General solvents and reagents were obtained from regular chemical supply houses and were used as received.

1-butanol (99% purity) and acetone-d₆ (99.5 atom% D) were purchased from Acros organics. Silicon dioxide (fused granular, 99.9% purity, mesh 4–20), sodium sulfate (99.0% purity), sodium hydroxide (≥97.0% purity), deuterated chloroform (99.8 atom% D), chromium acetylacetonate (97% purity), 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (95% purity), and deuterium oxide (99.9% atom% D) were purchased from Merck. Sulfuric acid (95–97% purity) and tetrahydrofuran (stabilized with BHT) were purchased from Boom B.V. Acetone (99.5% purity) and toluene were purchased from Macron Fine Chemicals. Endo-hydroxy-5-norbornene-2,3-dicarboximide was purchased from Fisher scientific. Walnut powder was bought from Brambleberry (Bellingham, WA, United States). Biomass preparation was performed via an earlier published procedure (Zijlstra et al., 2019b (link)), which includes ball-milling in order to obtain small particles and the removal of extractives from the biomass by a 2 h toluene extraction at reflux conditions in a roundbottom flask. The extraction liquor was removed by filtration and the extractive-free lignocellulosic feedstocks were dried in an oven in vacuo for 16 h at 70°C.