Sephadex lh 20 column

The fermentation broth CR17072 (8000 mL) was centrifuged and the fungus body was removed. The broth was extracted with ethyl acetate and added to a silica gel 60 (Merck KGaA, Darmstadt, Germany) and the active fractions were eluted with chloroform: methanol (10:1, v/v). Next, the active fractions were applied to a Sephadex LH-20 column (GE Healthcare, Pittsburgh, USA) and eluted with methanol. The active fractions were further purified by HPLC (Capcell Pak C18 UG, Shiseido, Tokyo, Japan, 20 × 250 mm; solvent, gradient elution with a mixture of water/acetonitrile, UV at 250 nm; flow rate, 10 mL/min). DNT and nine types of tryptoquivalines were obtained from a similar UV spectrum. The fractions were further concentrated under reduced pressure to provide pure compounds as white powders.

Nuclear magnetic resonance (NMR) was obtained on a Bruker AV-400 spectrometer (Bruker Corporation, Karlsruhe, Germany). HRMSESI data were recorded on a Q-Exactive Orbitrap MS system (Thermo Fisher Scientific, Bremen, Germany). UV data were obtained on an Evolution 220 UV-vis spectrophotometer (Thermo Fisher Scientific, Madison, United States). Infrared spectroscopy (IR) spectra were obtained on a Nicolet™ iS10 FTIR spectrometer (Thermo Fisher Scientific, Madison, United States). Optical rotations were recorded on an Autopol VI automatic polarimeter (PerkinElmer, Waltham, MA, United States). The silica gel (100–200 and 200–300 mesh, Qingdao Marine Chemical Factory, Qingdao, China) and Sephadex LH-20 column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) were used for open column chromatography (CC). Fractions were monitored by TLC (HSGF 254, Yantai Jiangyou Silica Gel Development Co., Yantai, China), and spots were visualized by heating silica gel plates after soaking in methanol supplemented with 10% H₂SO₄. The preparative HPLC was performed with UltiMate 300 HPLC (Thermo Fisher Scientific, Madison, United States) equipped with a YMC-Pack ODS-A column (250 × 10 mmI.D, S-5 μm, 12 nm, and flow speed = 2–3 ml/min, YMC Co., Ltd., Kyoto, Japan).

Dried roots of P. suffruticosa were purchased from a commercial oriental drug store, Human Herb, Gyoengsan, Korea. The organic solvents methanol (MeOH), n-hexane, ethyl acetate (EtOAc), dichloromethane (CH₂Cl₂), and butyl alcohol (BuOH) were obtained from Duksan Chemical (Anseong, Gyeonggi, Korea). The silica gel 60 (230–400 mesh ASTM, Merck, Darmstadt, Germany), ODS-A (12 nm, S-150 m, YMC, Tokyo, Japan), and Sephadex LH-20 column (GE Healthcare, Sweden) were used for open column chromatography. The NMR spectra were recorded on a Jeol ECA-500 spectrometer, operating at 500 MHz for ¹H and 125 MHz for ¹³C NMR spectra. The determination of the HPLC spectrum was recorded on an Agilent 1200 series (Agilent Technologies, Santa Clara, CA, USA) equipped with a photodiode array detector (PDA) and evaporative light scattering detector (ELSD).

Column chromatography was performed using a Sephadex LH-20 column (10–25 μm; GE Healthcare Bio-Science AB, Uppsala, Sweden) or MCI CHP 20P column (75–150 μm; Mitsubishi Chemical, Tokyo, Japan). Daisogel (40–60 μm; Daiso, Osaka, Japan) and Toyopearl HW-40F (30–60 μm; Tosoh Corp., Tokyo, Japan) were used in the stationary phase in a middle-pressure liquid chromatography (MPLC) system (Gilson, Seoul, Korea). For monitoring of each fraction, thin layer chromatography (TLC) was carried out using pre-coated silica gel 60 F₂₅₄ plates (Merck, Darmstadt, Germany) that were developed with chloroform, methanol and water (6:4:1 volume ratio), or benzene, ethylformate and formic acid (1:7:1 or 1:7:2 volume ratio). The spots were detected using ultraviolet radiation (254 nm) and by spraying with a FeCl₃ solution and 10% H₂SO₄ followed by heating. Structural identification was by nuclear magnetic resonance (NMR; Varian, Palo Alto, CA, USA), high resolution fast atom bombardment mass spectrum (HRFAB-MS; JEOL, Tokyo, Japan) and circular dichroism (CD; Jasco, Tokyo, Japan).

Grape seeds were obtained from berries of Chardonnay (C) and Pignoletto (P) after wine production (Faenza, Italy, 44°17′00″N 11°53′00″E) and they were air-dried at room temperature (final moisture 13%) and grounded to a granulometry of 2 mm. Briefly, according to Ky and Teissedre [27 (link)] 25 g of seeds were extracted with 500 mL of water-ethanol (3/7 v/v) under sonication for 40 min in an ultrasound bath (Starsonic 90 Liarre (Bologna, Italy) equipment with frequency 34 kHz) and the solvent was rotary evaporated under vacuum at 35 °C to remove ethanol. The resulting extracts were washed two times with 250 mL of n-hexane to remove lipid-soluble substances and then rotary evaporated to remove the residual hexane. After that, the extract was fractionated using Sephadex LH-20. The aqueous fraction was applied to a Sephadex LH-20 column (20 × 450 mm) (GE Healthcare, Barrington, IL, USA) and the resin was previously equilibrated with water (150 mL). The extracts were obtained using two water-ethanol ratio solutions: extracts F1 were obtained eluting with 300 mL of ethanol/water 80/20 v/v, whereas extracts F2 were eluted with 300 mL of ethanol/water 50/50 v/v. Both fractions were immediately frozen at −20 °C and then freeze-dried (Thermo HETO, power dry LYOLAB 3000; Waltham, MA, USA) and stored at −23 °C until the analysis.

The crude extract from 10 L culture of E264ΔBAC::Ptet/dis was fractionated with Sephadex LH-20 column (GE Healthcare) chromatography using MeOH as a mobile phase. Fractions containing F₂ (1) were combined and further purified by semipreparative reverse-phase HPLC (Agilent ZORBAX SB-C18 column, 250 × 9.4 mm, 5 μm; gradient elution 0–2 min, 64% ACN; 2–17 min, 64–90% ACN; 17–22 min, 95% ACN, 22–25 min, 64% ACN; 2.5 ml/min). Finally, 1.5 mg of disorazol F₂ (1) was obtained with retention time at 15.1 min.