7-hydroxycoumarin

Total photon counts at each pixel were normalized by the square of incident laser power to represent fluorescence intensity. Optical redox ratio was calculated by taking the ratio of mean fluorescence intensities within the segmented cytoplasm regions as: redox ratio = FAD/(NAD(P)H+FAD). This form has the advantage of being inherently bound between 0 and 1 and was established as a robust indicator of redox state in seminal studies performed by Chance40 (link). More recently, we have demonstrated that this optical redox ratio is strongly correlated to LC-MS/MS based assessments of both FAD/(NADH+FAD) and NAD⁺/(NADH+NAD⁺) concentration ratios18 (link)19 (link). NAD(P)H and LD fluorescence decay profiles were subject to a phasor transform as described by Stringari et al.23 (link). Fourier sine and cosine transforms mapped the time-resolved signal accumulated from 5-by-5 pixel bins about each image pixel to 2D phasor space (Supplementary Methods). 7-hydroxycoumarin fluorescence was used as a reference to correct for the instrumentation impulse response41 (link). Phasors from all images were accumulated by group to construct normalized density maps. For image segmentation, low-intensity regions (such as nuclei and other weakly fluorescent cellular compartments) were excluded via Otsu thresholding, while a combination of FAD intensity and NADH fluorescence lifetime was used to identify cytoplasm and lipid compartments in cells (see Supplementary Methods).

UPLC-MS analysis was performed following exact conditions described in Farrag, et al. [8 (link)]. Briefly, dried finely pulverized L. nudicaulis specimen (10 mg) was extracted by adding 2 mL 100% MeOH, containing 10 μg/mL⁻¹ umbelliferone as an internal standard sonicated for 20 min with frequent shaking, then centrifuged at 12,000× g for 10 min to remove debris. The filtered extract through a 22-μm filter (about 500 μL) was subjected to solid-phase extraction using a C₁₈ cartridge as previously described [114 (link)]. L. nudicaulis extract (2 μL) was loaded on HSS T3 column (100 × 1.0 mm, particle size 1.8 μm; Waters) installed on an ACQUITY UPLC system (Waters, Milford, MA, USA) equipped with a 6540 Ultra-High-Definition (UHD) Accurate-Mass Q-TOFLC/MS (Agilent, Palo Alto, CA, USA) coupled to an ESI interface, operated in positive or negative ion mode following conditions described in 113. Characterization of compounds was performed by the generation of the candidate formula with a mass accuracy limit of 10 ppm, and also considering RT, tandem MS2 data, and searching reference literature and the Phytochemical Dictionary of Natural Products Database. Peaks were detected in both negative and positive (deviating values in brackets) ion modes.

Acetaminophen (APAP) (4′-hydroxyacetanilide), N-acetyl-L-cysteine (NAC), 7-hydroxycoumarin (7-HC), and 7-methoxycoumarin (7-MC) were obtained from Tokyo Chemical Industry (Tokyo, Japan). 1-Phenyl-2-thiourea (PTU) was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were commercially available products of special reagent grade.

Commercial Luwak coffee was purchased from Bogor Indonesia as 100% pure, coarsely powdered coffee, ca. 2–4 mm in size, which has been heavily roasted and freeze-dried. It was compared to samples from two coffee-producing species: C. arabica, commonly known as arabica coffee, roasted arabica coffee (RCA), and green arabica coffee (GCA); the other is C. canephora var. robusta (known as green robusta coffee (GCC) or roasted robusta coffee (RCC)), collected from the Mina Gerais University Arboretum, Brazil, as entire seeds that were further powdered in a mortar using liquid nitrogen. Analysis was performed via NMR spectroscopy, ultra-performance liquid chromatography coupled with mass spectroscopy (UPLC-MS), and solid-phase microextraction coupled with the gas chromatography-mass spectrometry method (SPME/GC–MS). Samples subjected to SPME/GC-MS included Luwak coffee, roasted coffee, and roasted coffee blended with cardamom to comparatively evaluate the aroma profile. The NMR fingerprinting of coffee extracts was also conducted.
Freeze-dried coffee seeds were prepared for NMR analysis following the same protocol as used for herbal extracts [15 (link),16 (link),17 (link)]; about 150 mg of each coffee powder (n = 3) was homogenized with 6 mL of 100% MeOH containing 10 µg/mL umbelliferone (an internal standard for relative quantification using LC-MS), using an Ultra-Turrax (IKA, Staufen, Germany) at 11,000 rpm for 5 × 60 s, with 1-minute break intervals. The extract was vortexed for 1 min, centrifuged at 3000× g for 30 min, and then filtered. Afterward, 4 mL of the supernatant was aliquoted for NMR analysis and then dried in a stream of nitrogen. The dried extract was re-suspended with 800 μL of 100% methanol-d4, containing HMDS that has been adjusted to a final concentration of 0.94 mM. After centrifugation (13,000× g for 1 min), the supernatant was transferred to a 5-millimeter NMR tube for measurement. For the LC-MS analysis, 1 mL of the sample was aliquoted and placed on a 500 mg Octadecylsilane (C18) cartridge that was preconditioned with methanol. The samples were then eluted using 3 × 0.5 mL methanol; the eluent was then evaporated under a nitrogen stream and the obtained dry residue was resuspended in 1 mL of 100% methanol.

UPLC-MS acquisition was performed using ion-trap high-resolution testing under the same conditions as those used for coffee testing by El-Hawary et al. [13 (link)].
To profile the metabolites, 150 mg of each coffee powder specimen was homogenized with 5 mL MeOH (100% v/v) containing 10 µg/mL umbelliferone as an internal standard, using an Ultra-Turrax mixer (IKA, Staufen, Germany) adjusted at 11,000 rpm, mixed in five 20-second periods, with intervals of 1 min between each mixing period to guard against temperature increases and heating effects. The resulting suspensions were then vortexed vigorously, centrifuged at 3000× g for 30 min, and filtered through a 22 μm pore-size filter to remove plant debris. Then, 1 mL of the sample was aliquoted and pre-treated by placement on a 500 mg C₁₈ cartridge that was pre-conditioned with MeOH and Milli-Q water before elution; this was performed twice, using 3 mL of MeOH. Afterward, the eluent was evaporated under a nitrogen stream, and the obtained dry residue was re-suspended in 1 mL of MeOH.
The principal step of UPLC-ESI–HRMS analysis was conducted in triplicate (n = 3), with 2 μL introduced to a Dionex 3000 UPLC system (Thermo Fisher Scientific, Bremen, Germany), equipped with an HSS T3 column (100 × 1.0 mm, 1.8 μm; Waters^®; column temperature: 40 °C) and a photodiode array detector (PDA, Thermo Fisher Scientific, Bremen). The chromatographic conditions were optimized for improved peak elution, using a binary gradient elution protocol at a flow rate of 150 μL/min. The composition of the mobile phase varied between water/formic acid at 99.9/0.1 (v/v) (A) and acetonitrile/formic acid at 99.9/0.1 (v/v) (B). The protocol consisted of an isocratic step for 1 min with 5% mobile phase B, followed by a linear increase of B from 5% to 100% over 11 min. The mobile phase was kept isocratic for between 11 and 19 min at 100% B. After this, there was a return to 5% B within 1 min, and, finally, an additional 10 min, i.e., 20–30 min overall, for column re-equilibration using 5% B. The wavelength range of the PDA measurements used for detection was 190–600 nm.
The UPLC system was coupled with a high-resolution mass spectrometer, comprising an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a HESI electrospray ion source (spray voltage, positive ion mode 4 kV, negative ion mode 3 kV; source heater temperature, 250 °C; capillary temperature, 300 °C; FTMS resolution, 30,000). Nitrogen was used as both the sheath and auxiliary gas. The CID mass spectra (buffer gas: helium; FTMS resolution: 15,000) were recorded in a data-dependent acquisition mode (DDA) using normalized collision energy (NCE) of 35% and 45% The instrument was externally calibrated with Pierce^® LTQ Velos ESI positive ion calibration solution (product number 88323, Thermo Fisher Scientific, Rockford, IL, USA) and Pierce^® LTQ Velos ESI negative ion calibration solution (product number 88324, Thermo Fisher Scientific, Rockford, IL, USA).