Waters 600 pump

Reactions were performed in standard lab glassware when appropriate. Water was freshly distilled before use. All other solvents used were of HPLC grade quality. ESI mass spectrometry was performed by using a Waters (Manchester, UK) ZMD mass spectrometer equipped with a single quadrupole analyser. Samples were introduced to the mass spectrometer by flow injection using a Waters 600 pump (flow rate 0.1 mL min⁻¹ MeCN) and Waters 2700 autosampler. ¹H and ¹⁹F{¹H} NMR spectra were recorded in solution in deuterated H₂O or methanol on a Bruker DPX-400 or AV-400 spectrometers and are referenced to the residual solvent protons (¹H) and CCl₃F (¹⁹F) at 298 K. IR spectra were recorded neat (oils) or as Nujol mulls (solids) between CsI plates by using a Perkin–Elmer Spectrum 100 spectrometer over the range 4000–200 cm⁻¹. Microanalyses were undertaken by Stephen Boyer at London Metropolitan University. Compounds Bz(CH₂CO₂H)₂-tacn⋅HCl (H₂L⋅HCl)[5a ] and Li₂[Bz(CH₂CO₂)₂-tacn] (Li₂L)[9 ] were prepared by using the literature methods; Ga(NO₃)₃⋅9 H₂O and GaF₃⋅3 H₂O were obtained from Aldrich and used as received.

The n-butanol extract from leaves of L. japonica was reacted with DPPH by the same procedure as described in Section 3.4. The mixtures were passed through a 0.45 µm filter for HPLC analysis. A blank of aqueous extraction was used as a control. Analytical HPLC was carried out with a Waters 996 photodiode array detection (PDA), a Waters 600 Multisolvent Delivery, a Waters 600 system controller, and a Waters 600 pump. The separation was performed on a Shim-pack VP-ODS column (250 mm × 4.6 mm, i.d., 5 μm) (Shimadzu, Kyoto, Japan); gradient elution was performed using an A eluent (MeOH) and a B eluent (0.3% acetic acid in water, v/v) with the following linear gradient combinations: at t = 0, 70% B; at t = 20 min, 40% B; at t = 22.5 min, 40% B. Flow rate was 1.0 mL/min, and 10 μL portion was injected into the column. The effluent was monitored at 254 nm.

Individual specimens were dried in the shade at room temperature. Each dried sample was pulverized, and about 3 g power was weighed accurately and then extracted with 60 ml 100% methanol for 60 min in an ultrasonic bath, during which ice was added several times to keep the water temperature about 4 °C. After adding the loss weight, the extracts were filtered through a 0.45-μm membrane prior to HPLC analysis and determined within 24h. The chromatographic separation was performed on a Waters 600 HPLC system (Waters, USA), equipped with a Waters 2998 photodiode array detector, Waters 600 pump, Waters 2707 autosampler, and Waters in-line degasser AF. A Waters XTerra RP-18 column (4.6 × 250 mm, 5 μm) was also used. The column was kept at 35°C and the flow rate was 1 mL/min. The photodiode array detector was set at 245 nm. Methanol and H₂O (0.01 M triethylamine, pH 7.5) were used in the mobile phase according to a 55%:45% isocratic elution program.

Forsythiaside A, phillyrin and rutin were quantified by RP–HPLC. A Waters HPLC system equipped with Waters 600 pump, photodiode array detection (PDA) and Empower^TM 2 Chromatography Data Software (Waters Technologies, Milford, MA, USA) was used for quantitative analysis. Chromatographic separations were carried out using a Thermo ODS2 column (4.6 mm × 250 mm, 5 μm). The mobile phase consisted of acetonitrile (A) and water with 0.04% (v/v) phosphoric acid (B). Gradient elution was performed in a linear gradient according to the following program: 0–20 min, 15% A; 21–30 min, 25–30% A; 31–40 min, 30–55% A. The flow rate was 1.0 mL/min. UV detection wavelength was 330, 278 and 350 nm for forsythiaside A, phillyrin and rutin, respectively. Quantitative determination of the active compositions in the extracts was performed using external standards by means of a six-point calibration curve.

The weight‐average molecular weight (M_W) of chitosan was measured by gel permeation chromatography (GPC) (Li & Xia, 2010). The GPC equipment (Wyatt) consisted of an Ultrahydrogel 2000 (7.8 mm × 300 mm) combined with an Ultrahydrogel 250 (7.8 mm × 300 mm), an RI 150 refractive index detector, and a Waters 600 Pump. The eluent was 0.2 M CH₃COOH/0.15 M CH₃COONH₄. The temperature of the column and the flow rate were maintained at 303 K and 0.4 ml/min, respectively. Chitosan samples were prepared with the same acetate buffer at a concentration of 1 mg/ml, and dissolved samples were filtered through a 0.22‐μm filter. All data provided by the GPC system were collected and analyzed using the Waters Workstation software package.

Optical rotations were measured on a JASCO P-1020 polarimeter (10 cm cell) (American Laboratory Trading, East Lyme, CT, USA). Circular dichroism spectra were measured on a JASCO J-715 Spectropolarimeter Circular Dichroism/Optical Rotatory Dispersion (JASCO, Easton, MD, USA). UV spectra were recorded on a CAMSPEC M501 UV/Vis spectrophotometer (CAMSPEC, Sawston, UK). NMR spectra were recorded at 30 °C on a 800 MHz spectrometer. The ¹H and ¹³C chemical shifts were referenced to the DMSO-d₆ solvent peaks at δ_H 2.50 and δ_C 39.52 ppm. Standard parameters were used for the 2D NMR spectra obtained, which included gCOSY, gHSQCA, gHMBCAD, NOESY, and ROESY. For HPLC isolation, a Waters 600 pump equipped with a Waters 966 PDA detector (Waters, Milford, MA, USA) and Gilson 715 liquid handler were used (Gilson, Lewis Center, Delaware County, OH, USA). An Onyx C18 column (5 μm, 21.2 mm × 150 mm) and Hypersil BDS C18 column (5 μm, 10 mm × 250 mm) were used for semi-preparative HPLC. A Phenomenex Luna C18 column (3 μm, 4.6 mm × 50 mm) was used for LCMS controlled by MassLynx 4.1 software (Waters, Milford, MA, USA). All solvents used for extraction and chromatography were HPLC grade purchased from RCI Labscan or Burdick & Jackson (Lomb Scientific, Sydney, Australia), and the H₂O used was ultrapure water (Arium^® proVF, Sartorius Stedim Biotech, New York, NY, USA).

The dried stems of M. tenacissima (10 kg) were crushed and extracted with 95% ethanol (30 L × 4) at r. t. to give 987 g of extract (MTE), and each extraction time is 16-24h. The extracts were filtered and concentrated, followed by re-suspended in 2 L distilled water and partitioned with ethyl acetate (EtOAc) (0.5 L × 4) to furnish an EtOAc-soluble residue (570 g) after removing the solvent. The residue was subject to column chromatography on silica gel (9 × 80 cm) eluting with gradient petroleum ether and EtOAc (6:11:1) and petroleum ether and ethyl acetate (3:1) to afford eight fractions FR1-FR8. FR5 was analyzed by HPLC (Agilent 1200 Series, Agilent Technologies, USA) on a YMC Triart C18 column (250 × 4.6 mm, 5 μm, YMC CO., LTD., Japan) eluted with 80 % (v/v) methanol in water at a flow rate of 1.0 mL/min and detected at 210 nm. Compounds 1-5 were isolated from FR5 on preparative HPLC systems equipped with a Waters 600 pump, a Waters 2487 UV detector, a YMC-Pack R & D ODS-A C18 column (250 × 20 mm, 5 μm, YMC CO., LTD., Japan) and an N 2000 chromatography workstation. Samples of FR5 were eluted with 78 % (v/v) methanol in water at a flow rate of 4.0 mL/min and detected at 210 nm. The structure the five compounds were elucidated on the basis of nuclear magnetic resonance (NMR) analyses and comparison with the literatures.