Chloroform

PDMS-DMS-B12 [(carboxydecyl)-terminated
polydimethylsiloxane, 15–30
cSt] and PDMS-DMS-B25 [(carboxydecyl)-terminated polydimethylsiloxane,
450–550 cSt] were purchased from Gelest and a vinyl-terminated
PDMS (100 cSt) from abcr. Dichloromethane (CH₂Cl₂) and DMF of HPLC grade were obtained from Fisher Chemical, LiBr
was obtained from Alfa Aesar, and chloroform, diethyl ether (Et₂O), THF, MgSO₄, and DMF were purchased from VWR.
3-(4,5-Dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
was bought from Calbiochem, Dulbecco’s modified Eagle medium
(DMEM) and fetal bovine serum (FBS) were purchased from Gibco, DMF-d₇ was purchased from Deutero, and Aerosil R
106 was purchased from Evonik. N,N-Dimethylacetamide
(DMAc), 2-isopropenyl-2-oxazoline (iPOx), CuCl, CuCl₂,
tris(2-pyridylmethyl)amine (TPMA), 2-chloropropionitrile (CPN), 2,2′-azobis(2-methylpropionitrile)
(AIBN), and thioglycolic acid were purchased from Sigma-Aldrich. iPOx
was distilled under reduced pressure prior to RDRP. For FRP, the monomer
was used as received. Dialysis was performed with a no.6 dialysis
tubing from Spectrum Laboratories with a molecular weight cutoff (MWCO)
of 1 kDa.
Size exclusion chromatography (SEC) was carried out
on two different
instruments. On the one hand, a Viscothek GPCmax instrument from Malvern,
equipped with a PFG column from Polymer Standard Service GmbH (300
× 8 mm, 5 μm particle size) and run with DMF containing
10 mM LiBr as the mobile phase, was used. The samples were prepared
as a 10 mg*mL^–1 solution, filtered through a 0.2
μm PTFE syringe filter prior to injection, and eluted at a flow
rate of 0.75 mL*min^–1 at 60 °C. The system
was calibrated with polystyrene standards from the Polymer Standard
Service GmbH using a conventional calibration of the refractive index
detector. On the other hand, an SEC system consisting of Shimadzu
LC–20, a Shimadzu refractive index detector, and two PPS PFG
5 μm columns or PSS GRAM 5 μm columns from Polymer Standard
Service GmbH (300 mm × 8 mm) at 25 °C were employed. DMAc
with an addition of 0.1 wt % LiBr was used as an eluent at a flow
rate of 1 mL*min^–1, and samples were injected with
a concentration of 1 mg*mL^–1. Poly(methyl methacrylate)
standards from Polymer Standard Services GmbH were used for calibration.
Infrared (IR) spectra were recorded with a PerkinElmer 100 Series
FTIR spectrometer equipped with ATR using a scan number of 128 as
well as on a Nicolet 8700 FTIR spectrometer from Thermo scientific
equipped with a Nicolet Continuum microscope and a germanium ATR crystal
using 64 scans and a resolution of 4 cm^–1.
Nuclear magnetic resonance (NMR) measurements were performed on
a Bruker Avance III 300 MHz spectrometer in deuterated solvents at
25 °C, referenced to the respective internal solvent signal.
XPS signals were recorded using a Thermo Scientific Nexsa G2 surface
analysis system equipped with a microfocused, monochromatic Al Ka
X-ray source (1486.68 eV). An X-ray beam of 400 mm size was used.
The spectra were acquired in the constant analyzer energy mode with
a pass energy of 200 eV for the survey. Narrow regions were collected
using a pass energy of 50 eV. Charge compensation was achieved with
the system dual beam flood gun. The Thermo Scientific Avantage software,
version 6.7.0, was used for digital acquisition and data processing.
Spectral calibration was determined by using the automated calibration
routine and the internal Au, Ag, and Cu standards supplied with the
K-Alpha system. The surface compositions (atomic %) were determined
by considering the integrated peak areas of the detected atoms and
the respective sensitivity factors. The fractional concentration of
a particular element A was computed using where I_n and s_n are the
integrated peak areas and the Scofield sensitivity factors corrected
for the analyzer transmission, respectively.
The elastomer samples
for rheological characterization and gel
fractions were cured in a circular Teflon mold in a drying and heating
chamber from Binder at 120 °C. Rheological characterizations
were conducted on an Anton Paar MCR 502 rheometer with a plate–plate
geometry (8, 10, and 25 mm diameters) through stress-controlled oscillatory
tests at 24 °C. Amplitude sweeps were performed with a constant
frequency of 1 rad*s^–1 and an increasing strain
rate γ from 10^–3 % to 10° % for E-B12
and 10^–2 % to 10² % for E-B25 and E-Syl.
Subsequently, frequency sweeps at a constant rate of deformation of
γ = 1 × 10^–2 % for E-B12 and of γ
= 1% for E-B25 and E-Syl were conducted. The reaction kinetics was
directly investigated by measuring the freshly prepared formulations
on the heated rheometer plate at the respective temperatures (room
temperature, 80, 120, and 160 °C). The gel fractions of the elastomer
pellets were determined by washing the pellets with CH₂Cl₂ over 24 h. After separation of the solid fraction
by decanting or filtration and subsequent drying on air at RT for
48 h, the weight loss of pellets was determined and the gel fraction
was calculated. Tensile tests were performed on a TA Instruments DMA
Q800 with an increase in the applied normal force of 0.25 N per 60
s and a sample geometry of width = 40 mm, length = 100 mm, and thickness
= 2 mm.
Cytotoxicity of elastomers was tested using extracts
according
to ISO 10993 and direct contact cytotoxicity. Extracts from two types
of elastomers were obtained by extraction into growth medium (DMEM
+ 10% FBS) for 24 h. The extract thus obtained was labeled as 100%
and was subsequently diluted with growth medium to 50, 10, and 1%
solutions. The pure extracts and their dilutions were then added to
3T3 fibroblasts that grew on a 96-well tissue culture plate at a concentration
of 5 × 10³ cells per well. After 24 h of incubation,
quantification of living cells was performed using the MTT test. Extracts
from wells were replaced by 100 μL of MTT at a concentration
of 0.5 mg*mL^–1 in growth media (DMEM + 10% FBS)
and incubated for 3 h. Afterward, MTT was removed, and 100 μL
of dimethyl sulfoxide was added to the wells to extract the water-insoluble
formazan formed by living cells. The absorbance was measured via a
plate reader at 595 nm (Multiscan FC, Thermofisher). For contact cytotoxicity,
cells were seeded in the 24-well tissue culture plate at a concentration
of 50 × 104 cells per well and incubated overnight. The next
day, pieces of elastomers covering 10% of the well surface were placed
in triplicate into the cell containing wells and incubated for 24
h, followed by an MTT assay as described above.

The following chemicals and reagents were used in the present study: ethanol (≥99.93%), sodium hydroxide (0.1 mol/L or 0.1 N), chloroform (≥99%), and cyclohexane (≥99.8%) were supplied by VWR (VWR International, LLC, 1–3 Rue d’Aurion, Rosny-sous-Bois, France). Glacial acetic acid with ≥99% purity was supplied by Fisher Scientific Ltd. (Loughborough, UK). Diethyl ether (≥99.8%) was purchased from Honeywell Riedel-de Haen GmbH, Seelze, Germany. Potassium iodide (≥99.0%), phosphoric acid (H₃PO₄) (49–51%), and sodium thiosulfate (Na₂S₂O₃.5H₂O) with 99% purity were purchased from Sigma-Aldrich (Saint Louis, MO, USA). HPLC-grade syringic acid ≥97%, and tyrosol (2-(4-hydroxyphenyl) ethanol) with ≥98% purity were supplied by Sigma-Aldrich Chemie GmbH (Steinheim, Germany). LC-MS-grade methanol with ≥99.9% purity and acetonitrile with 100% purity were supplied by Fisher Scientific Ltd. (Loughborough, UK). Ultrapure water was obtained from a MilliQ system (Millipore, Bedford, MA, USA).
For sensory analyses, watch glasses, odorless markers to mark the tasting glasses, tasting sheets, bottled water, apples, and standard cups were used following the standard procedure of the International Olive Council [31 ]; additionally, an air oven was used (Super M 1072, Barcelona, Spain) to maintain the temperature of the sample at 28 ± 2 °C before the analysis. Moreover, a balance (Sartorius AG120S, Göttingen, Germany) with a precision of +0.1 g, a thermometer for controlling the room temperature, and a thermometric probe (J. P. SELECTA, Barcelona, Spain) to measure the temperature of the samples were used.

The main standards used in the analyses of phenolic compounds by LC–MS/MS, and all other relevant data were presented in detail in a previous work [9 (link)]. The additional standards were leontopodic acid A, cat. N 6026S, and leontopodic acid B, cat. N 6032S.
Chemicals applied for TPC, antioxidant activity assays (ATBS and DPPH): ethanol HPLC grade (Panreac, Barcelona, Spain), gallic acid, Folin–Ciocalteu reagent 2 N, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), DPPH• (2,2-diphenyl-1-picrylhydrazyl) (Sigma-Aldrich, St. Louis, MO, USA), sodium carbonate (Merck, Darmstadt, Germany), potassium persulfate and absolute ethanol (Neon, Suzano, SP, Brazil), and quercetin dihydrate (Sigma-Aldrich Chemie GmbH., Steinheim, Germany).
Chemicals used for the GC–FID analyses: Supelco 37 Component FAME Mix (CRM47885), toluene (pure, VWR International, France), sulfuric acid (98%, MerckKGaA, Germany), sodium chloride (pure, MerkKGaA, Darmstadt, Germany), potassium bicarbonate (pure, VWR International, Paris, France), chloroform (99.8% VWR International, Paris, France), sodium sulfate (pure, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany), and helium (99.9999%, Air Liquide A/S).
The rest of the reagents applied were of the highest purity: methanol ≥ 99.9%, ethanol ≥ 99.8% and n-hexane ≥ 99% were purchased from Honeywell Riedel-de-Haen (Seelze, Germany), ethyl acetate ≥ 99.5% from JLS-Chemie Handel GmbH (Hannover, Germany), methyl tert-butyl ether ≥ 99.8%, and acetonitrile ≥ 99.9% from Sigma-Aldrich (Darmstadt, Germany) and bone dry grade CO₂ (99.99% pure; No water, Messer, Sofia, Bulgaria).

Cellular RNA was isolated using RNAiso Plus (TAKARA, Kusatsu, Japan), according to the manufacturer’s instructions. Briefly, the cell pellets were resuspended in 1 mL of RNAiso Plus by pipetting, mixed with 0.1 mL chloroform (Amresco, Cleveland, OH, USA), and centrifuged at 12,000× g for 15 min at 4 °C. The supernatants were mixed with 0.25 mL 100% isopropanol, and isolated RNA pellets were washed with 70% ethanol and centrifuged at 7500× g for 5 min. Dried pellets were dissolved in 30 µL diethyl pyrocarbonate (DEPC) water, and RNA was quantified while using a Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was prepared from RNA using a cDNA synthesis kit (PrimeScript™, TAKARA). qRT-PCR was performed to determine the RNA levels. The primer was mixed with distilled water and then placed in 384-wells. Template (cDNA) and SYBR green (TAKARA) were subsequently added, and then validated using PCR machine (Bio-Rad, Hercules, CA, USA). Table S1 lists the genes of interest.