Agilent 1260 instrument

Optical rotations were measured on a polAAr3005 polarimeter, and UV data were recorded on a Shimadzu Biospec-1601 spectrophotometer. The CD spectra were recorded on a JASCO J-815 spectropolarimeter. IR data were recorded using a Nicolet Magna-IR 750 spectrophotometer. ¹H and ¹³C NMR data were acquired with Varian Mercury-400, -500, and -600 spectrometers using the solvent signals (acetone-d₆: δ_H 2.05/δ_C 29.8, 206.1; DMSO-d₆: δ_H 2.49/δ_C 39.5; CDCl₃: δ_H 7.26/δ_C 76.7) as references. The HSQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. ESIMS and HRESIMS data were obtained using an Agilent Accurate-Mass-Q-TOF LC/MS 6520 instrument equipped with an electrospray ionization (ESI) source. The fragmentor and capillary voltages were kept at 125 and 3500 V, respectively. Nitrogen was supplied as the nebulizing and drying gas. The temperature of the drying gas was set at 300 °C. The flow rate of the drying gas and the pressure of the nebulizer were 10 L/min and 10 psi, respectively. All MS experiments were performed in positive ion mode. Full-scan spectra were acquired over a scan range of m/z 100–1000 at 1.03 spectra/s. HPLC separations were performed on an Agilent 1260 instrument (Agilent, USA) equipped with a variable wavelength UV detector. Chiral HPLC analysis and separation were performed on a Kromasil 5-CelluCoat RP column (4.6 × 250 mm; 5 μm; AkzoNobel).

Polysaccharide is a kind of macromolecular carbohydrate, which is consisted of at least 10 monosaccharides. The composed of monosaccharides is a critical element of polysaccharides, which is closely related to bioactivities of polysaccharides. The composition of ASP was analyzed by PMP‐label method as previously reported (Jin et al., 2018). In brief, the sample was hydrolyzed with trifluoroacetic acid (TFA) at 100°C for 6 hr. Then, the hydrolyzed production was reacted with PMP at 70°C for 30 min. The standards, including Glu, D‐Glu, Xyl, Rha, Man, Ara, Gal, Fuc, and lactose (internal reference), were treated in parallel with the sample. After several extractions, the aqueous phase was filtered into 0.45 μm membrane and separated by HPLC on an Agilent 1260 instrument (Agilent), which was equipped with Agilent ZORBAX ODS column (5 μm, 4.6 mm × 150 mm) plus a gradient elution program (Table 1). The A phase consisted of 0.1% methanoic acid and 50 mM ammonium acetate, while the B phase was acetonitrile and C phase was 0.1% methanoic acid. All the mobile phases were eluted in the column at a flow rate of 0.6 ml/min. The separation was recorded under 254 nm wavelength. The identifications were based on retention time with respect to standards. Quantification was carried out by integration of the chromatographic peak area.

Glucose, fructose, and sucrose were determined using the method described by Xu et al. [53 (link)]. A 0.5 g sample was homogenized with 4.0 mL ultrapure water and incubated at 80 °C for 15 min. The supernatant was collected and centrifuged at 8000× g for 5 min at room temperature. The resulting solution was filtered through a 0.45 um cellulose acetate filter using an Agilent 1260 instrument equipped with a refractive index detector and a CNW NH2-RP column (4.6 mm × 250 mm, 5 μm, Anpu Technology Co., Ltd., Shanghai, China). The mobile phase consisted of a mixture of acetonitrile and water (80:20, v/v), with a flow rate of 1 mL/min. The column temperature was 25 °C, while the detector temperature was set at 40 °C. An injection volume of 10 uL was used for the analysis. HPLC-grade standard products were purchased from Sigma (USA).

HO, used as starting material, was isolated from Cephalotaxus fortunei Hook. f. by our group, and its structure was identified by ESI-MS and NMR. All reagents were purchased from Energy Chemical (Shanghai, China) and Aladdin (Shanghai, China) and were used without any further purification. Thin-layer chromatography (TLC) was performed using silica gel plates (GF254, Qingdao Marine Chemical Ltd., Qingdao, China) and visualized under ultraviolet (UV) light (254 nm). Silica gel column chromatography was performed using 200–300 mesh (Qingdao Marine Chemical Ltd., Qingdao, China).
NMR spectra were recorded on Bruker Avance III-500 and Bruker Avance III-600 spectrometers (Bruker, Karlsruhe, Germany) at ambient temperature using TMS as the internal standard. High-resolution electrospray ionization (HRESI) mass spectra were carried out using an Agilent 6520B Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). All final compounds have purity >95% (Figure S2) as determined by an Agilent 1260 instrument equipped with VWD detector (Agilent Technologies, Santa Clara, CA, USA) using a COSMOSIL 5C18-AR-II column (250 mm × 4.6 mm, i.d., 5 μm, Nacalai Tesque, Kyoto, Japan).

For the analysis of samples obtained by the method described above for TLC, reverse-phase high-performance liquid chromatography was used for product separation and mass spectrometry for more accurate identification of possible modifications of fluorescent dyes that occurred in the cell.
The Agilent 1260 instrument (Agilent Technologies, Santa Clara, CA) equipped with a PDA detector and a mass spectrometric detector, and a Zorbax SB-C8 column of 5 µm (150 mm × 4.6 mm; Agilent Technologies) with an acetonitrile gradient were used. In the experiments, electrospray ionization was employed. Only substances that fluoresce at wavelengths greater than 500 nm when excited with a wavelength of 488 nm were analyzed. The results were processed in MassLynx and ChemDoodle software.