Sephadex lh 20

The microbial extract library preparation and screening platform for EPI activity is discussed in Supplementary Material. The 48 h old inoculum of soil isolate IMTB 2501 was grown in R-2A broth (Himedia^®, India), supplemented with 1% NaCl, and 0.2% calcium carbonate. After 96 h of incubation, cells were removed by centrifugation and extract was prepared using Diaion^® HP-20 resin. The crude extract was fractionated on Sephadex^TM LH-20 (GE Healthcare) column using a step gradient of methanol: water. The active fractions were pooled and processed further by reverse-phase high-pressure liquid chromatography (RP-HPLC) (Waters, XBridge^® BEH C18 OBD^TM Prep Column 130 Å, 5 μm, 10 mm × 250 mm). The mobile phase consisted of solvent A as 10 mM ammonium acetate (pH 5.5) and solvent B as 100% acetonitrile. The flow rate was kept constant at 3 mL/min. The following gradient was used; 5% solvent B for 5 min, 5 to 95% B in 45 min, hold at 95% B for 10 min. The peaks were collected and assayed for the bioactivity. The active peak (termed as RP2) was identified and subjected to lyophilization. The purified compound was subjected to gas chromatography (Varian 450-GC) coupled with mass spectrometry (Varian 20-MS). The spectrum was recorded in an electron-ionization mode. NMR spectra (¹H, ¹³C, DEPT, COSY, and HSQC) were recorded on a Bruker 400 MHz spectrometer in DMSO-d₆.

Labdanum absolute was subjected to fractionation by molecular-sizing column chromatography as described by Vogt and Gülz [66 (link)] and used in recent works [3 (link),10 (link),12 (link)]. Absolute was directly eluted on Sephadex^TM LH-20 (GE Healthcare Biosciences AB) compacted in a 25 cm long and 1.5 cm width column using methanol as eluent. Fractions of about 2–3 mL were continuously collected and analyzed by thin-layer chromatography using silica gel TLC plates (F254s 0.2 mm 10 × 20 cm) (Merck, Darmstadt, Germany), as described by Vogt and Gülz [66 (link)]. Toluene: ethyl acetate (9:1) was used as eluent and, after drying, plates were visualized at 312 nm using a transilluminator to detect flavonoid bands. To detect diterpene bands, plates were sprayed with 10 M H₂SO₄ solution and heated at 100 °C for 10 min in a ventilated chamber. Purple/red bands were assigned to the diterpenes and yellow bands to the flavonoids because they matched the bands visualized at 312 nm. Fractions presenting purple bands were joined as the diterpene fraction (Dit) and fractions presenting flavonoid bands were joined as the flavonoid fraction (Flv). This procedure was conducted for the three independent labdanum absolutes.

UV spectra were obtained by a Milton Roy Spectronic 3000 Array spectrophotometer (Rochester, NY, USA). Optical rotations were measured on a JASCO P-2000 polarimeter (Kyoto, Japan). CD spectra were recorded using a Jasco J-815 CD spectrophotometer (Kyoto, Japan). IR spectra were recorded on a Nicolet^TM iS50 FT-IR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) and Perkin Elmer FT-IR 1760X spectrometer (Boston, MA, USA). HR-ESI spectra were measured on a Bruker APEX II mass spectrometer (Karlsruche, Germany) and Agilent 6540 UHD Accurate-Mass Q-TOF mass spectrometer (CA, USA). NMR was recorded on a Bruker Advance NEO 400 MHz NMR spectrometer (Karlsruche, Germany) and a Varian VNMRS-600 spectrometer (Lexington, MA, USA). Medium-performance liquid chromatography (MPLC) and flash column chromatography (Flash CC) were performed by using a PuriFlash^® XS 420 (Advion Inc., NY, USA), Sepacore^® purification system (Buchi AG, Flawill, Switzerland), or a ceramic pump (VSP-3050; EYELA, Kyoto, Japan). Silica gel 60 (70–230 or 230–400 mesh ASTM, Merck, Darmstadt, Germany), LiChroprep^® RP-18 (25–40 μm, Merck, Darmstadt, Germany), and Sephadex^TM LH-20 (GE Healthcare, Amersham, UK) were used as a stationary phase material for column chromatography (CC). Organic solvents (commercial grade) were redistilled prior to their use as a mobile phase composition.

The ¹H and ¹³C, DEPT, HSQC, HMBC, ROESY, and COSY NMR spectra were recorded on a Avance 500 MHz instrument (Bruker, Switzerland). The high-resolution electrospray ionization mass spectra (HR-ESI-MS) were obtained from an Agilent 6530 Accurate-Mass Q-TOF LC/MS system (Agilent Technologies Co., Ltd., Waldbronn, Germany). Semi-preparative HPLC was performed on a Agilent 1100 series system,)using an ODS column (Zorbax SB-C₁₈, 9.4 × 250 mm, 5 µm, Agilent Technologies Inc., Santa Clara, CA, USA). Column chromatography was performed on silica gel (200–300 mesh and 300–400 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), Sephadex^TM LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), and octadecyl silica (ODS, 50 μm, YMC Co., Ltd., Tokyo, Japan). Thin-layer chromatography was performed on a precoated GF₂₅₄ plate (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), RP-18 F₂₅₄S plates (Merck, Kenilworth, NJ, USA) and compounds were visualized by spraying with aqueous 10% H₂SO₄ and then heating for 3–5 min.

Optical rotations were measured with a Jasco P1000 digital polarimeter. UV spectra were recorded by a Hewlett Packard 8453 UV–vis spectrometer. CD spectra were obtained with a Jasco J-715 circular dichroism spectrophotometer. 1D and 2D NMR were performed on a VNS 600 MHz spectrometer operating at 600 MHz for protons and 150 MHz for carbon. Chemical shifts are expressed in ppm and referenced relative to the residual solvent signals. Mass spectra were recorded with a Micromass LCT mass spectrometer, and the lock mass calibration was applied to accurately measure masses. Semi-preparative HPLC was performed with an Agilent system consisting of a vacuum degasser, quaternary pump, diode array detector (DAD), and Luna 5u C₁₈ (2) 100 Å column (250 × 10.00 mm; Phenomenex). Column chromatography (CC) was carried out on either silica gel (Merck KGaA, 70–230 mesh) or Sephadex^TM LH-20 (GE Healthcare Sweden). TLC was performed with precoated silica gel 60 F254 plates (Merck KGaA) and spots were visualized under UV light (254 and 365 nm) or by spraying with 20% H₂SO₄ and heating.