Modular hplc system

The applied analytical method was based on the description of Li et al. [35 (link)] with slight modifications. The extracts were analyzed by the modular HPLC system (Shimadzu, Japan) equipped with a fluorescence detector, which was controlled by ClassVP 6.2 software. The peaks were detected by an UV detector at a wavelength of 436 nm. The mobile phase consisted of water containing 20% methanol (A) and acetonitrile containing 10% methanol (B) and both were supplemented with 0.5% trifluoroacetic acid (Sigma). Separations were performed on a Gemini 250 × 4.6 mm, 5 μm reversed phase column (Phenomenex, Torrance, CA) coupled with Phenomenex C18 guard column with a flow rate of 1 mL/min using a gradient program started with 10% B, and reached to 70% B until 10 min, to 90% until 15 min and to 25 min until 100%, which was kept until 60 min and reduced to initial eluent ratio and held to pressure stabilization. The injection volume was 5 μL. The calibration was done with serial dilution of hypericin and emodin standards (Sigma) in the range of 250 μg/mL to 7.8 μg/mL based on the retention times of hypericin (32.8 min) and emodin (16.9 min). The quantity of hypericin and emodin present in the samples were quantified using the equations y = 0.000142788 x—5.07 and y = 0.0000808111 x—4.66, respectively, while the r values were 0.998 and 0.999 for hypericin and emodin, respectively.

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.

Isolation and purification of GAG chains from the cell cultures were performed^{67 (link)}. Cells were homogenized with ice-cold acetone, extracted with ice-cold acetone three times, and air-dried thoroughly. The delipidated acetone powder was digested with actinase E (one-tenth the weight of acetone powder) in 0.1 M borate-sodium, pH 8.0, containing 10 mM CaCl₂ at 55 °C for 48 h. The samples were adjusted to 5% v/v in trichloroacetic acid and centrifuged. The GAG-containing materials were precipitated from the resultant supernatants by mixing with ethanol, dissolved in water, and subjected to gel filtration on a PD-10 desalting column (Cytiva) using water as an eluent. The flow-through fractions were collected and evaporated to dryness. The purified GAG fraction containing the CS and HS chains was digested with 5 mIU of chondroitinase ABC (Seikagaku), or a mixture of 0.5 mIU of heparinase (Seikagaku) and 0.5 mIU of heparitinase (Seikagaku), at 37 °C for 3 h. The digests were derivatized with the fluorophore 2-AB and then analyzed by anion-exchange HPLC using a YMC-Pack PA-G column (4.6 × 250 mm, YMC, Kyoto, Japan). A modular HPLC system (Shimadzu) was operated by LabSolutions LC/GC (Ver. 5.42, Shimadzu). The identification and quantification of the resulting disaccharides were achieved by comparison with authentic unsaturated CS and HS disaccharides (Seikagaku).