Breeze 2

The nuclear magnetic resonance (NMR) spectra of all materials was recorded with an NMR spectrometer (AVANCE III HD 600 MHz, Bruker, Rheinstetten, Germany). The molecular weights of poly(l-lactic-co-glycolic acid) (P(L)LGA) were measured by Gel Permeation Chromatography (GPC, Breeze2, Waters, Milford, MA). The rheological curves of P(L)LGA, PEG, and GEM·HCl mixtures were determined by a rheometer (DHR-2, TA, New Castle, DE). The electrospray ionization (ESI) mass spectrum of GEM·HCl was determined by a mass spectrometer (QExactive, Thermo Fisher Scientific, Waltham, MA). The drug loading (DL) and drug release behavior of all microdevices were measured by a microplate reader (Nivo, PerkinElmer, Waltham, MA). The surface and section morphology of microdevices were observed by a scanning electron microscope (SEM, JSM-IT200, Japan Electronics, Minato, Japan). All histological sections of mice were recorded with inverted fluorescence microscope (IFM, TE2000, Nikon, Minato, Japan). Blood routine tests were measured by an automatic hematology analyzer (BC-2800vet, Mindray, Shenzhen, China). Blood biochemical parameters were determined by an automatic biochemical analyzer (Chemray 800, Rayto, Shenzhen, China).

To test the esterification ability of GMGL, reactions using glycerol and linolenic acid as substrate were conducted. The reaction mixture in a flask contained 0.64 g linolenic acid, 1.36 g glycerol, 20 mg lyophilization enzyme of GMGL WT and buffer (20 mM Tris-HCl, 20 mM NaCl, pH 8.0, (2% w/w, with respect to total reaction mixture). The flask was incubated in a glycerol bath of 40 °C having a magnet stick with a speed of 500 rpm. The samples from the reaction were withdrawn at periodic intervals and analyzed with HPLC. Peak-area percentages were obtained by the Waters Breeze 2 software using an area normalization method. The MAG content was calculated as the percentage of MAG peak area vs. the sum of peak area of MAG and fatty acid.

Macromolecular peptidoglycan was re-suspended in 500 μl of 50 mM phosphate buffer pH 4.9 and digested with Cellosyl (Hoechst AG) 100 μg/ml final concentration at 37 °C overnight. The enzyme reaction was stopped by boiling the sample for 15 min in a water bath. Coagulated protein and insoluble contamination were eliminated by centrifuging in a MiniSpin® Plus (Eppendorf) at 14,500 rpm for 15 min at room temperature. Muropeptides contained in the soluble fraction were mixed with 1/3 volume of 0.5 M sodium borate buffer (pH 9.0) and reduced with excess sodium borohydride (NaBH₄) for 30 min at room temperature. The excess borohydride was neutralised with phosphoric acid (dilution 1:10) to pH 3–4. Finally the samples were filtered through Millex®-GV Filter 0.22 μm (Millipore) units and stored at −20 °C. Reduced muropeptides were separated and analysed by HPLC (Breeze™ 2 System, Waters). Elution products were detected at wavelength 204 nm and identified by the retention time obtained. The relative quantity of muropeptides present in each sample was determined by integration of their respective absorption areas (Breeze™ 2, Waters) and expressed as a molar fraction (mol%) of the total content. When required, the individual peaks were collected, vacuum dried and stored at −20 °C.

The molecular weights of lignins were characterized by a gel permeation chromatography instrument (Waters Breeze 2) equipped with a refractive index detector (Waters model 2414) and two Waters Styragel columns (models: HR 0.5 and HR 2; dimensions: 7.8 × 300 mm)^{22 (link)}. Polystyrenes with different molecular weights were used as standard compounds. Tetrahydrofuran was used as the mobile phase at a flow rate of 1.0 ml min⁻¹. The column compartment and the refractive index detector temperature were set at 40 °C. To facilitate dissolution of lignins in tetrahydrofuran, hydrogenolysis of lignins and hot-pressed lignins was conducted before gel permeation chromatography analysis.

The identifications of AND, NAD, DDA were performed according to conventional approach for known structure: crystal form (Optical microscope, Shanghai Guangmi instrument Co. Ltd, in P.R. China, XSP-2C), Melting point(Yuhua instrument Co. Ltd. in P.R. China, Micro melting point apparatus X-4), IR ( PerkinElmer, American, Spectrum-100), UV (SHIMADZU,Japan, UV-2450 ) and HPLC (Waters ChemStation, Breeze2, USA).
The quantitative study of AND, NAD, and DDA in Andrographis paniculata and extracts from its processing by-products was performed by HPLC (Figure 4). The HPLC system (Waters ChemStation, Breeze2, USA) consisted of a low-pressure binary pump (model Waters 1525), a UV detector (model Waters 2489) and an autosampler (model Waters 2700 Autosampler) with a 48-vial capacity sample. The separations were carried out on a Phenomenex RP-18 column with a particle size of 250 mm (5 μm). A Phenomenex RP select B guard column with a particle size of 5 μm was placed in front of the analytical column. The chromatographic conditions were as follows: filtered (22 μm) acetonitrile and water, gradient programmed isocratic elution, room temperature, run time of 50 min, injection volume of 10 μl, and wavelength of 225 nm.Figure 4

HPLC chromatograms of three diterpenoids in Andrographis paniculata. A. Three standard diterpenoids; B. Blank sample; and C. Sample of AEE.

A total of 500 μL serum was combined with 100 μL of 6M NaOH at 60 °C for 40 min. For acidification, hydrolyzed serums were mixed with 35% perchloric acid and centrifuged for 10 min at 37 °C. Subsequently, for 10 min, 250 μL of supernatant was incorporated with 25 μL of 2,4-dinitrophenylhydrazine (DNPH). A serum sample that had been derivatized was analyzed using an HPLC (Waters Breeze-2, USA) with an ODS2 reverse-phase column. The mobile phase was 38: 62 acetonitrile: 0.2% acetic acid HPLC-grade water. MDA concentration in serums was measured at 310 nm using HPLC under isocratic conditions [29 ].