Shim pack gist

Chromatography was performed on a modular HPLC system from Shimadzu (Kyoto, Japan); it contained a system controller (CBM-20A), two Nexera X2 pumps, a degasser (DGU-20ASR), and a column oven (CTO-20AC). Automated extractions were carried out with a DBS-MS 500 (CAMAG, Muttenz, Switzerland). Analytes were separated on a Shim-pack GIST (4.6 × 50 mm, 5 μm STEAROYL, 227-30017-3) analytical column (Shimadzu, Kyoto, Japan). A filter frit (KrudKatcher Ultra, Phenomenex, Torrance, CA, USA) was connected upstream to the analytical column. Mobile phase A consisted of water plus 0.1% formic acid and 2 mM ammonia fluoride, while methanol supplemented with 0.1% formic acid and 2 mM ammonia fluoride was used as mobile phase B. The following stepwise gradient was applied: 40% A (0–1.0 min), 40–90% A (1.0–2.0 min), 90% A (2.0–3.0 min), and 40% A (3.01–4.0 min). The flow rate was set at 1.0 mL/min at 40 °C. The HPLC liquid stream was connected to an 8060 tandem mass spectrometer (Shimadzu, Kyoto, Japan). The mass transitions and compound specific settings were included in Table S1.

Chromatographic quantification of the pomegranate compounds was performed by high-performance liquid chromatography using a Shimadzu system (Shimadzu Corporation, Kyoto, Japan) consisting of a pump (LC-20AT), diode array detector (SPD-M20A), system controller (CBM-20A), autoinjector (SIL-20A), LC-20AT quaternary pump, and Shimadzu LC solution software. The chromatographic separation and the ellagic acid and punicalagin determination were performed using a reverse-phase Shimadzu Shim-Pack GIST analytical column C18 (100 mm × 4.6 mm × 3 μm) at 30 °C, as described by Santiago et. al, with some modifications [80 ]. The mobile phase consisted of acetonitrile (phase B) and water containing 5% formic acid (v/v) (phase A) using the following gradient program: 0–5 min, 97–95% A; 5–10 min, 95–85% A; 10-16 min, 85-70% A; 16–18 min, 70–97% A; 18–25 min, 97% A. The flow rate was 0.8 mL min⁻¹. Peaks were determined by comparison with an authenticated ellagic acid (Sigma-Aldrich, St. Louis, MO, USA) and punicalagin α/β (Sigma-Aldrich) standard. PPE samples were diluted in methanol, homogenized in an ultrasonic bath for 30 min, and then filtered (0.45 µL). The injection volume was 5 μL, and we used a wavelength of 260 nm. All samples were prepared in triplicate.

Calibration curves for each standard were made using six concentrations (3.125 to 100 μg/mL). GE and GG03 were filtered through 0.22 μm membrane filters (Woongki Science Co., Ltd., Seoul, Korea) and a 10 μL aliquot of each extract solution in 80% MeOH (10.0 mg/mL) was injected into the HPLC system. Formic acid was purchased from Sigma. HPLC-grade acetonitrile and water were obtained from Honeywell Burdick and Jackson Inc. (Muskegon, MI, USA). HPLC analysis was achieved using a Waters 600S (Waters, Milford, MA, USA) with a Waters 2487 UV detector (254 nm). The column was a Shimpack Gist (4.6 × 250 mm, particle size: 3 µm, Shimadzu Co., Kyoto, Japan). The mobile phase consisted of 0.1% Formic acid in water (solvent A) and acetonitrile (solvent B), which were eluted at a flow rate of 0.4 mL/min with the following gradient elution with concentration of solvent 30% (0.01 min), 30% (5 min), 55% (10 min), 55% (13 min), 80% (20 min), 80% (23 min), 100% (30 min), 100% (60 min). The quantitative analysis was replicated three times.

HPLC-PDA was employed to profile the phytochemical composition of the SLT extract used in the study. The HPLC-PDA analysis was performed on a Prominence-I LC-2030 3D Plus Shimadzu HPLC system controlled by Lab Solutions software (Shimadzu, Australia). Separation was achieved using a Shimadzu Shim-pack GIST (Shimadzu, Australia) reverse phase C18 column (4.6 × 150 mm I.D., 5 μm) maintained at 40°C.
The SLT extract was dissolved by sonication in 30% aqueous acetonitrile for 30 min at 5 mg/ml. Individual solutions of standards, crocin, ginsenoside Re, ginsenoside Rg1, ginsenoside Rd, quercetin, kaempferol, and isorhamnetin were prepared to 1 mg/10 ml in 30% aqueous acetonitrile for identification and combined for analysis. The sample and mixed standard were syringe-filtered with 0.45 μm PTFE.
The SLT HPLC-PDA profiles were generated by 20 μl injection. The mobile phase consisted of 0.1% (v/v) aqueous formic acid (mobile phase A) and 0.1% (v/v) formic acid in acetonitrile (mobile phase B). The gradient program was 10% B for 1 min with a linear increase, to 45% B at 45 min, and then, a wash and re-equilibration. The mobile phase flow rate was maintained at 1.1 ml/min. The PDA was set to acquire absorbance data from 190 to 800 nm.

LC/MS/MS analyses were conducted using
a Shimadzu LC/MS system equipped with a binary pump (Nexera X2, LC-3AD),
degasser (DGU-20A5R), autosampler (Nexera X2, SIL-30AC), column oven
(CTO-20AC), and a triple quadrupole mass analyzer (LCMS-8045). A Phenyl
Group C18 Column (Shim-pack GIST, 2 μm, 2.1 mm × 100 mm,
Shimadzu, Japan) was used to separate different polar analytes under
40 °C. The mobile phase (A) consisted of 50 mM ammonium acetate
in deionized water, while the mobile phase (B) consisted of pure methanol.
During HPLC analysis, the injection volume and flow rate of mobile
phase was set at 1 μL and 0.3 mL/min, and the eluted gradient
of the organic mobile phase (B) was 0.0–1.0 min: 60%, 1.0–10.0
min: 60–100%, 10.0–15.0 min: 100%, 15.0–16.5
min: 100–60%, and 16.5–23.0 min: 60%. The operational
parameters for the mass spectrometer were set at an interface voltage
of 4.0 kV, nebulizer gas flow rate of 3 L/min, heating gas flow rate
of 10 L/min, interface temperature of 300 °C, DL temperature
of 250 °C, drying gas flow rate of 10 L/min, and heat block temperature
of 400 °C.