Cbm30a

Petunia corollas were collected, freeze-dried and ground to powder, and 0.1 g of the powder was placed in 1.0 mL of 70% aqueous methanol at 4 °C overnight. The extract was then centrifuged at 10,000g for 10 min, and the supernatant was filtered through a microporous membrane (0.22-μm pore size) for liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis.
The metabolites were analysed by ultra-performance liquid chromatography (UPLC) (Shim-pack UFLC SHIMADZU CBM30A, http://www.shimadzu.com.cn/) and MS/MS (AB SCIEX 6500 QTRAP) under the following conditions described by Li and Song^{46 (link)}: column, water ACQUITY UPLC HSS T3 C18 1.8 μm, 2.1 × 100 mm; mobile phase, the aqueous phase was ultrapure water (0.04% acetic acid), and the organic phase was acetonitrile (0.04% acetic acid); water/acetonitrile gradient, 95:5 V/V for 0 min, 5:95 V/V for 11.0 min, 5:95 V/V for 12.0 min, 95:5 V/V for 12.1 min, and 95:5 V/V for 15.0 min; flow rate, 0.4 mL/min; column temperature, 40 °C; and injection volume, 2 μL. The electrospray ionization (ESI) temperature was 500 °C, the MS voltage was 5,500 V, the curtain gas (CUR) was 25 psi, and the collision-induced dissociation (CAD) parameter was set to high. In triple quadrupole mode (QQQ), each ion pair was scanned for detection based on the optimized decompression potential (DP) and collision energy (CE)^{47 (link)}.

The lipid metabolites from testicular contents were determined via UPLC–MS/MS. In short, the UPLC (Shim‐pack UPLC Shimadzu CBM30A) and MS/MS (QTRAP 6500+) system was configured with a resolution of 30,000 in order to acquire UPLC–MS/MS data. The analysis was conducted in positive ion mode using a spray voltage of 5.5 kV and in negative ion mode using a spray voltage of −4.5 kV, while maintaining a capillary temperature of 500°C. The mass spectrometer was set to scan 50–1500 m/z. The nitrogen sheath and nitrogen auxiliary gas were adjusted to flow at rates of 30 and 10 L/min, respectively. Solvent A consisted of 0.04% acetic acid (Fisher Scientific) diluted with water (Millipore) in a volume‐to‐volume ratio, while solvent B was prepared by combining 0.04% acetic acid (Fisher Scientific) with acetonitrile (Fisher Scientific) in a volume‐to‐volume ratio. The rate of gradient flow was adjusted to 0.4 mL/min, while maintaining a column temperature of 40°C. The linear gradient was as follows: 5% B at 0 min, 95% B at 11.0 min, 95% B at 12.0 min, 5% B at 12.1 min, and 5% B at 14 min. To guarantee system stability, the QC samples were injected four times initially. For all analyses, a Waters ACQUITY UPLC HSS T3 C18 column measuring 100 mm × 2.1 mm with a particle size of 1.8 μm was utilized. The contents of lipid metabolites were detected using MetWare (http://www.metware.cn/).

The data acquisition instrument system mainly includes Ultra Performance Liquid Chromatography (UPLC) (Shim-pack UFLC SHIMADZU CBM30A, http://www.shimadzu.com.cn/) and Tandem mass spectrometry (MS/MS) (Applied Biosystems 4500 QTRAP, http://www.appliedbiosystems.com.cn/).
UPLC was run under following conditions: (1) Waters ACQUITY UPLC HSS T3 C18 1.8 µm, 2.1 mm × 100 mm; (2) The aqueous phase was ultra-pure water (0.04% acetic acid is added) and the organic phase was acetonitrile (adding 0.04% acetic acid); (3) Elution gradients: 95:5 v/v at 0 min, 5:95 v/v at 11.0 min, 12 min, 12.1 min and 15 min; (4) The flow rate was 0.4 mL/min, the column temperature was 40 °C, and the injection volume was 5 μL. MS/MS worked with these conditions: (1) Curtain gas (CUR) was set to 25 psi; (2) Electrospray ionization (ESI) was set to 550 °C; (3) the MS voltage was set to 5500V; (4) Dclusteringotential (DP) was optimized; (5) Collision energy (CE) was optimized; (6) Collision-activated dissociation (CAD) was set to high.

We used UPLC (Shim-pack UFLC SHIMADZU CBM30A, http://www.shimadzu.com.cn/ (accessed on 29 August 2019)) and MS/MS (Applied Biosystems 6500 QTRAP, Waltham, MA, USA, http://www.appliedbiosystems.com.cn/ (accessed on 29 August 2019)) to analyze the sample extracts. The UPLC analysis was performed under the following conditions. UPLC: column, Waters (Shanghai, China) ACQUITY UPLC HSS T3 C18 (1.8 μm, 2.1 mm × 100 mm); solvent system, water (0.04% acetic acid): acetonitrile (0.04% acetic acid); gradient elution program, 95:5 V/V at 0 min, 5:95 V/V at 11.0 min, 5:95 V/V at 12.0 min, 95:5 V/V at 12.1 min and 95:5 V/V at 15.0 min; flow rate, 0.40 mL/min; column temperature, 40 °C, and an injection volume of 2 μL. The separated samples were connected to electrospray ionization (ESI)-QTRAP-MS for mass spectrometric analysis.

The collected petunia corollas were freeze-dried and ground to powder, and 0.1 g of powder was placed in 1.0 ml of 70% aqueous methanol at 4 °C and stored overnight. Then, the extract was centrifuged at 10 000 g for 10 min, and the supernatant was filtered through a microporous membrane (0.22 μm pore size) for analysis.
The metabolites were analysed by ultra-performance liquid chromatography (UPLC) (Shim-pack UFLC SHIMADZU CBM30A, http://www.shimadzu.com.cn/) and tandem mass spectrometry (MS/MS) (AB SCIEX 6500 QTRAP) under the conditions described by Li and Song (2019) (link). In the triple quadrupole, each ion pair was scanned for detection based on the optimized decompression potential and collision energy (Chen et al., 2013 (link)). The qualitative and quantitative MS analysis of metabolites in the samples was performed on metabolites based on the self-built Metware database (MWDB) (Metware, Wuhan, China), and multiple reaction monitoring (MRM) (Chen et al., 2013 (link)).

Ripe noni fruit were provided by noni fruit planting base in Wuzhishan City, Hainan Province, China. After oven drying at 65 °C, the noni slices were ground into powder with the aid of an electric pulverizer (CH-200A, Chenhe Shengfeng Industry and Trade Co., Ltd., Yongkang, China) to pass a 1 mm screen. The noni powdered fruit were extracted with distilled water (1:25 ratio of raw material to water, w/v) by soaking for 12 h at 70 °C. Then, the filtrate after filtration (using filter paper with a maximum pore size of 15~20 μm) was concentrated to one-tenth of the volume under reduced pressure in a rotary evaporator at 65 °C. The concentrated solution was mixed with absolute ethanol (1:4, v/v) and then left to stand at 4 °C for 48 h to precipitate the polysaccharides. The polysaccharide-rich extract of noni fruit (NFP) was obtained by centrifugation (1200× g, 15 min) and lyophilized using a freeze dryer (Biosafer-10 C, Safer (China) Co., Ltd., Nanjing, China). The yield of NFP was 11.02%. We used ultraperformance liquid chromatography (UPLC; Shim-pack UFLC Shimadzu CBM30A)–electrospray ionization tandem mass spectrometry (MS, Applied Biosystems 6500 Q TRAP, UPLC-ESI-MS/MS) system to identify the structures of the active ingredients of NFP, according to the method by Li et al. [25 (link)]. The results are shown in Table 1.

In total, 100 mg powder was dissolved in 1.0 mL Buffer A (70% aqueous methanol). Samples were incubated overnight at 4 °C, and then centrifugated at 10,000× g for 10 min. The supernatant was filtrated using 0.22 μm pore size (SCAA-104, ANPEL, Shanghai, China). Then, HPLC-MS/MS analysis was performed LC-ESI-MS/MS consisting of Shim-pack UFLC SHIMADZU CBM30A coupled to 6500 Q TRAP.