Agilent 1100

The hairy roots were dried at 50 °C in an oven until a constant weight was obtained and then grounded into powder form. The powder was then extracted with chromatographic pure methanol for 60 min under sonication. Next, the methanol extract was applied to the high-performance liquid chromatography (HPLC) system-Agilent-1100-for analysis. The Agilent-1100 was carried out with a H₂O (containing 0.5% HCOOH) (A)/acetonitrile (B) gradient by a ZORBAX SB-C₁₈ chromatographic column (250 × 4.6 mm, 5 μm) at 30 °C [6 (link)]. The reference standards of dihydrotanshinone I (DT-I), tanshinone I (T-I), cryptotanshinone (CT), and tanshinone IIA (T-IIA) were purchased from the Chengdu Mansite Pharmaceutical Co. Ltd. (Chengdu, China). In addition, dihydrotanshinone I (0.0025, 0.0050, 0.0100, 0.0200, and 0.0400 mg/mL), tanshinone I (0.002, 0.004, 0.006, 0.008, and 0.010 mg/mL), cryptotanshinone (0.001, 0.005, 0.010, 0.025, 0.050, and 0.100 mg/mL), and tanshinone IIA (0.0005, 0.001, 0.002, 0.004, 0.006, and 0.008 mg/mL) were used to prepare the standards curves. The methanol extract of hairy roots was analyzed and the peaks identified and contrasted in comparison with the available standards.

The nucleotides (GMP, IMP, XMP, AMP, Hx, HxR) in samples were determined according to Zhang, Y. et al. [2 (link)] with some modifications. An Agilent 1100 high performance liquid chromatograph (HPLC, Agilent Company, Santa Clara, CA, USA) equipped with Hypersil ODS2−C18 (4.6 mm × 250 mm, 5.0 μm) and an ultraviolet (UV) detector was used. A total of 20 g of the crushed muscle powder was placed in a 50 mL plastic centrifuge tube and mixed with 40 mL of 5% trichloroacetic acid (TCA) solution. The mixture was placed in a 4 °C refrigerator for 2 h before it was centrifuged at the speed of 2725× g for 10 min. The supernatant was transferred into a 100 mL beaker and its pH was adjusted to 6.5 with 3 mol/l KOH solution. The solution was filtered with a qualitative filter paper (Fushun Mingzheng Filter Paper Factory, Anhui, China) and diluted to 50 mL with deionized water. The diluted solution was filtered with a 0.45 μm organic membrane (Shanghai Xinya Purification Equipment Co., Ltd., Shanghai, China) and 10 μL of it was injected into the Agilent 1100 HPLC. The optimized gradient chromatographic conditions were as follows: mobile phase A (pure methanol) 2–15%, B (KH₂PO₄ buffer (98–85%)) is 0.05 mol/L; column temperature: 30 °C; flow rate: 0.8 mL/min. The nucleotide contents were calculated using the external standard method.

Samples were analysed using SEC (injection volume 10 µL) with Agilent 1100. The column used was a TSKgel G3000SWXI (7.8 × 300 mm) from TOSOH Bioscience with a running buffer composed of 100 mmol L^‐1 sodium phosphate (monobasic), 400 mmol L^‐1 sodium chloride, pH 6.7. The flow rate was 1.0 mL min^‐1 and protein was detected using UV detectors at 214 nm and 280 nm.
For SEC combined with fluorescence detection (Agilent 1100) with the dyes, injection of samples was increased to 50 µL to strengthen the signal. The same amount of dye used in the plate assay was added to the samples before injection into the column. Samples containing SYPRO Orange were excited at 495 nm and the emission read at 590 nm. Samples containing ProteoStat were excited at 530 nm and emission read at 605 nm.

Quantification of sugars, volatile fatty acids and alcohols were performed with high-performance liquid chromatography (HPLC) system (Agilent 1100), consisting of a G1310A isocratic pump, a G1313A ALS autosampler, a Transgenomic ICSep ICE-ION-300 column, a G1316A column thermostat set at 45 °C and a G1362A RID refractive index detector, measuring at 45 °C (all modules were from Agilent 1100 (Agilent Technologies, CA, USA). The measurement was performed with 0.005 mol L⁻¹ H₂SO₄ as solvent, with a flow rate of 0.325 mL min⁻¹ and a pressure of 48–49 bar. The injection volume was 40 µL.

With the use of High Performance Liquid Chromatography (HPLC) system (Agilent 1100), which consists of two LC pumps, a UV/Vis detector, and a C18 column (250 mm × 4.6 mm, 5 µm), flavonoid components from the methanol extracts of the four treated C. sativum plants were identified. With an isocratic elution (70:30) program, the mobile phase consisted of acetonitrile (A) and 0.2% (v/v) aqueous formic acid (B). The detection wavelength was set at360 nm^{70 (link)}.
Using HPLC, The phenolic and flavonoid components from Acacia saligna FAE were identified by HPLC analysis^{70 (link)}. For instance, to analyze the phenolic compound, HPLC (Agilent 1100, Agilent ChemStation) had a UV/Vis detector, two LC pumps, and a C18 column (125 mm × 4.6 mm, 5 µm particle size) was used to gather and examine chromatograms. By using a gradient mobile phase of two solvents—Solvent A (Methanol) and Solvent B [Acetic acid in water (1:25)], phenolic compounds were isolated. For the first 3 min, the gradient program was maintained at a concentration of 100% B. The concentration of eluent A was then raised to 80% for the following 2 min, then decreased to 50% once again for the following 5 min detection wavelength at 250 nm. This was followed by 5 min of 50% eluent A. As a result, the order of phenolic compounds was established utilizing this mobile phase to verify standard compounds.

Optical rotations were measured with a Rudolph Autopol III polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). Shimadzu UV-2550 spectrometer (Beckman, Brea, CA, USA) was used for scanning UV spectroscopy. IR spectra were obtained on a Tensor 27 spectrometer, as KBr pellets (Thermo, Pittsburgh, PA, USA). NMR spectra were recorded on an AV-500 spectrometer (Bruker, Bremen, Germany) with TMS (Tetramethylsilane) as an internal standard. HR-ESI-MS were performed on an API QSTAR Pulsar mass spectrometer (Billerica, MA, USA). Silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), RP-18 (40–70 mm, Fuji Silysia Chemical Ltd., Kasugai Aichi, Japan) and Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) were used for column chromatography (CC). Semipreparative HPLC (Agilent 1100, Agilent Technologies Inc., Santa Clara, CA, USA) was performed on an Agilent 1100 liquid chromatograph with a Zorbax SB-C₁₈, 9.4 mm × 25 cm, column. Fractions were monitored by TLC and spots were visualized by heating after spraying with 5% H₂SO₄ in ethanol.