Na2co3

Cells were lysed on ice in 550 µl (six-well plates) or 1 ml (10 cm dishes) of RIPA buffer (Boston Bioproducts) in presence of cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail (Millipore Sigma) and PhosSTOP (Millipore Sigma) for 10 min. Lysates were cleared by centrifugation at 16,000 × g for 10 min at 4 °C. 10% of the lysed cells was set aside as input. set aside as input. Pull down and washes were performed as in ref. ^{27 (link)}. Briefly, lysates were mixed with Pierce Streptavidin Magnetic Beads (Thermo Fisher) at a ratio of 100 µl beads/4 million cells. Lysates were incubated with beads with constant rotation for one hour at room temperature and then overnight (O/N) at 4 °C. Beads were then applied to a magnet and subjected to the following washes: two times with 1 ml of RIPA, one time with 1 M 1 ml of KCl (Thermo Fisher), one time with 1 ml of 0.1 M Na₂CO₃ (VWR), one time with 1 ml of freshly prepared 2 M Urea (VWR) resuspended in 10 mM Tris-HCl pH 8.0 (Thermo Fisher) and two times with RIPA. Proteins were eluted from beads by adding 150 µl (six-well plates) or 500 µl (10 cm dish) of non-reducing Laemmli (Boston bioproducts) containing 20 mM DTT (Thermo Fisher) and 2 mM biotin (Cayman chemicals) and boiled for 20 min. Input was diluted with 2X sample buffer (Sigma). For mass spectrometry, beads were processed as follows.

A batch of oil resin extract (157 g) from commercial tamanu oil (provided by “Laboratoire de Cosmétologie du Pacifique Sud” manufacture) from French Polynesia was first partitioned with EtOH (provided by Fisher chemical) and an aqueous alkaline solution of Na₂CO₃ (from VWR chemicals) (10%, v/v). Its organic fraction was washed with distilled water and then dried with MgSO₄ (provided by VWR chemicals) to give a neutral fraction (53 g) after solvent evaporation. This fraction was submitted to flash liquid chromatography on an open column with a silica gel (240–300 mesh) using a stepwise gradient from cyclohexane (provided by VWR chemicals) to EtOAc (provided by VWR chemicals), yielding 12 fractions. Fractions having similar R_f values on silica gel TLC (cyclohexane-acetone, both provided by VWR chemicals, 60:40, v/v) were combined. Fractions 7, 9 and 11 were submitted to repeated preparative HPLC using a Varian Dynamax Si column (250 × 21.4 mm id, five μm with cyclohexane-EtOAc (10:90) in isocratic eluent conditions. This chromatographic purification network led to the isolation of new compounds tamanolides E1 and E2 as a mixture (two mg) besides standard known compounds namely, calophyllolide, inophyllums (C, D, E, P), calanolides (Gut 70 and A, 12-oxo-calanolide) and tamanolides (D, P).

Nitrate and nitrite reference material (1000 mg L⁻¹), certified for ion chromatography, were obtained from Sigma-Aldrich (Stenheim, Germany). Na₂CO₃ (>99.5%) was supplied by VWR International s.r.l. (Milan, Italy). Ultrapure water (18.2 MΩ-cm), produced using the Arium^® mini essential UV system (Sartorius AG, Goettingen, Germany), was used for preparing both mobile phase samples and standard solutions. The calibration curves of nitrite and nitrate were obtained by injecting the following concentrations of both compounds: 0.01, 0.1, 1.0, 5.0 and 10.0 mg L⁻¹.

A spruce sulfite pulp with DP 590 from Domsjö fabriker AB, Domsjö/Sweden, N-methylmorpholine-N-oxide of BASF/Leverkusen/Germany as a 50 wt.% aqueous solution, and polyacrylate from Evonik/Essen/Germany were used to produce the SAP fiber. SAP is partly crosslinked with NaOH, containing 3.43 mmol COO⁻/g (Titration) and 19.9% Na (ICP-OES). Glucose, AgNO₃, NaCl, CuSO₄·5H₂O, and Na₂CO₃ were purchased from VWR International GmbH/Darmstadt/Germany as p.a. products.

The spectrophotometric assay was conducted as previously described (Awosika and Aluko, 2019 (link)) with some modifications. Root extracts were diluted with ultrapure water (Purelab Flex, ELGA Veolia, United Kingdom) to give concentrations of 10 µg/ml to 1,000 μg/ml. Of these dilutions, 15 µl were mixed in 96-well microtest plates (VWR, Darmstadt, Germany) with 105 µl of 0.1 M phosphate buffer, pH 6.8 and 15 µl of α-glucosidase (0.5 U/ml) from Saccharomyces cerevisiae (Sigma-Aldrich, Taufkirchen, Germany). Following 5 min pre-incubation at 37°C, 15 µl 10 mM p-nitrophenyl-α-D-glucopyranoside (Sigma-Aldrich, Taufkirchen, Germany) in the same buffer were added as a substrate to initiate the reaction. The mixture was incubated for 20 min at 37°C, before 50 µl 2 M Na₂CO₃ (VWR, Darmstadt, Germany) were added to stop the reaction. A microplate reader (iEMS Reader MF, MTX Lab Systems, Helsinki, Finland) was used to measure the absorbance at 405 nm. Acarbose (Sigma-Aldrich, Taufkirchen, Germany) served as a reference inhibitor. The percentage inhibition of α-glucosidase was calculated by using the following equation: $Inhibition(\%)= \frac{\left[\left(AbC-Ab{C}_{blank}\right)-\left(AbS-Ab{S}_{blank}\right)\right]}{\left(AbC-Ab{C}_{blank}\right)}\times 100$ AbC, absorbance of the control; AbS, absorbance of the sample. IC₅₀ values were calculated by nonlinear regression using GraphPad Prism version 8.1.1.