QFY was prepared by mixing the dried aqueous extracts of seven herbal ingredients: BZ, 1.6 g; DG, 3 g; GC, 0.6 g; RS, 1.2 g; SD, 1.8 g; SZR, 1.2 g; and YZ, 1 g. Following extraction with distilled water at 80°C for 30 min, the mixture was cooled to room temperature and centrifuged at 13,000 rpm for 10 min. The supernatant was recovered and precipitated with ethanol at the final concentrations of 50%, 75%, and 90% overnight at 4°C. After centrifugation at 6,000 rpm for 30 min, the supernatant was collected to yield the corresponding ethanol solutions of QFY. The ethanolic materials were dried with a rotary evaporator, dissolved in DMSO, and sterilized by passing through a 0.22 μm syringe filter for bioassays. For HPLC separation, the compounds were separated on an ACE C18 HPLC column (250 × 4.6 mm, 5 μm) from Advanced Chromatography Technologies Ltd. (Aberdeen, Scotland, UK) under the control of the Waters Controller 600S HPLC system coupled with a photodiode array detector (Waters, Milford, MA, USA). The column temperature was maintained at 25°C. The mobile phases of (A) methanol and (B) 0.1% aqueous formic acid were pumped into the column at a flow rate of 1.0 mL/min to form the gradient as follows: 0-25 min, 5-50% A; 25-45 min, 50-70% A; 45-47 min, 70-95% A; and 47-53 min, 95% A. The fractions were collected, dried, and dissolved in DMSO for subsequent bioassays.
Photodiode array detector
Manufactured by Waters Corporation
Sourced in United States, Australia, Belgium
The Photodiode array detector is a laboratory instrument used for the detection and analysis of various compounds. It operates by directing a beam of light through a sample and capturing the resulting light pattern using an array of photodiode sensors. The photodiode array detector provides rapid, sensitive, and precise measurements of the light absorption or emission characteristics of the sample.
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Lab products found in correlation
Acquity uplc system, by Waters Corporation (3 mentions)
Empower 3, by Waters Corporation (3 mentions)
Xbridge c18 column, by Waters Corporation (3 mentions)
Empower software, by Waters Corporation (3 mentions)
Waters acquity uplc system, by Waters Corporation (2 mentions)
Acquity uplc beh c18 column, by Waters Corporation (2 mentions)
Hplc system, by Waters Corporation (2 mentions)
Lycopene, by Merck Group (2 mentions)
β carotene, by Merck Group (2 mentions)
C18 reverse phase hplc guard and analytical column, by Phenomenex (2 mentions)
Haemin and ucb, by Frontier Scientific (2 mentions)
Xbridge beh 125 sec 3.5 m hplc column, by Waters Corporation (2 mentions)
Sunfire c18 column, by Waters Corporation (2 mentions)
Waters alliance e2695 hplc system, by Waters Corporation (2 mentions)
L9879, by Merck Group (2 mentions)
Acquity hss t3 75 2.1 mm 1 8 μm column, by Waters Corporation (2 mentions)
Acquity hss t3 1.8 μm vanguard precolumn, by Waters Corporation (2 mentions)
Gemini 400, by Agilent Technologies (1 mentions)
Q tof premier mass spectrometer, by Waters Corporation (1 mentions)
Quattro premier xe, by Waters Corporation (1 mentions)
57 protocols using photodiode array detector
1
Extraction and Purification of Multiherb QFY
2
Analytical Characterization of Chemical Compounds
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Analytical grade chemical reagents were purchased from Chengdu Changzheng Chemical Factory (Sichuan, People’s Republic of China). Thin-layer chromatography was performed on 0.20 mm silica gel 60 F254 plates (Qingdao Ocean Chemical Factory, QingDao, People’s Republic of China). Proton nuclear magnetic resonance (1H NMR) spectra were recorded at 400 MHz on a Varian spectrometer model Gemini 400 and reported in parts per million (ppm). Chemical shifts (δ) are quoted in ppm relative to tetramethylsilane as internal standard, where (δ) tetramethylsilane =0.00 ppm. The multiplicity of the signal is indicated as s, singlet; d, doublet; t, triplet; q, quartet; and m, multiplet, defined as all multipeak signals where overlap or complex coupling of signals makes definitive descriptions of peaks difficult. Mass spectra were obtained using a Q-TOF Premier mass spectrometer utilizing electrospray ionization (Waters Quattro Premier XE, Milford, MA, USA). The purity of compounds was determined to be ≥97% by high-performance liquid chromatography analysis with a photodiode array detector (Waters, Milford, MA, USA) and an Atlantis C18 chromatographic column (150×4.6 mm2, id 5 μm; Waters).
3
Carbohydrate Separation and Quantification
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Carbohydrates were separated by ion-pairing reversed-phase chromatography on an HSS T3 UPLC column (2.1 × 150 mm, 1.8 µm; Waters) with a linear gradient of 10 mM TEAB, (pH 8.5) from 2% acetonitrile to 90% acetonitrile (containing 10 mM TEAB) over 7 min at 45°C and a flow rate of 0.4 ml/min. Eluted compounds were detected by UV absorbance (photodiode array detector; Waters) and by ESI-MS detection (QDa; Waters) or MALDI-TOF MSMS.
The QDa was operated in an electrospray negative ion mode by applying a voltage of 0.8 kV. The cone voltage was set at 15 V. The probe temperature was set at 600°C. A full mass spectrum between m/z 100 and 1200 was acquired at a sampling rate of 8.0 points/s.
For quantification of ADP heptose, calibration standard solutions at 7 concentrations from 10 to 100 pmol/µl were analyzed in single ion recording (SIR) mode at m/z 618. The calibration curves were established by plotting the peak areas (m/z 618) against the concentrations of analytes with linear regression analysis.
The QDa was operated in an electrospray negative ion mode by applying a voltage of 0.8 kV. The cone voltage was set at 15 V. The probe temperature was set at 600°C. A full mass spectrum between m/z 100 and 1200 was acquired at a sampling rate of 8.0 points/s.
For quantification of ADP heptose, calibration standard solutions at 7 concentrations from 10 to 100 pmol/µl were analyzed in single ion recording (SIR) mode at m/z 618. The calibration curves were established by plotting the peak areas (m/z 618) against the concentrations of analytes with linear regression analysis.
4
Quantitative Analysis of Cs-4 Extract
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Cs-4 extract, which is a commercial health product, was manufactured and supplied by Royal Medic Group Limited (Hong Kong SAR, China), with the contents of adenine, adenosine, and cordycepin being determined by UPLC-UV quantitative analysis. Briefly, Cs-4 powder was extracted by 25 mL of a mixture of water and ethanol (1:1, v/v). The mixture was sonicated for 30 min and then centrifuged at 3000× g at room temperature. The resultant supernatant was concentrated to 5 mL by rotary evaporation at 60 °C. The concentrated extract was reconstituted to a volume of 25 mL and gave a final concentration of 8 mg/mL. The Cs-4 extract (2 μL) was separated using a Waters ACQUITY UPLC system coupled with a photodiode array detector (Waters Corporation, Milford, MA, USA). A mobile phase consisting of water (solvent A) and acetonitrile (solvent B) was applied for programmed gradient elution with varying concentrations of A and B with time (0 min: 100% A and 0% B, 10 min: 97% A and 3% B, 18 min: 80% A and 20% B, and 21–24 min: 100% A) on a Waters ACQUITY UPLC BEH C18 column (130 Å, 1.7 μm, 2.1 mm × 100 mm) (Waters Corporation, Milford, MA, USA) at a flow rate of 0.25 mL/min and a temperature of 40 °C. The detection wavelength was set at 261 nm. The contents of adenine, adenosine, and cordycepin in the Cs-4 extract fraction were determined using a calibration curve.
5
UPLC Analysis of Phenolic Compounds
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Phenolic compounds and organic acids were identified using Ultra-Performance Liquid Chromatography (ACQUITY UPLC H-Class System, Waters Corporation, Milford, MA, USA). The separation was archived on an Acquity UPLC BEH C18 column (2.1 mm, 150 mm, 1.7 µm, Waters) thermostated at 35 °C. The flow rate of 0.4 mL.min−1 and the gradient elution with water and acetonitrile (both containing 0.1% formic acid, pH = 2) were used. The identification of peaks was based on a comparison with the retention times of chemical standards. The detection was performed in a Waters Photodiode Array Detector set at λ = 280 nm and λ = 320 nm using an external standard.
6
Carotenoid Extraction and Quantification
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Carotenoids were extracted following the method described by Liu et al. with slight modifications (Liu et al., 2007 (link)). Carotenoids were determined on a reverse phase Analytical YMC Carotenoid Column C30 (150 × 4.6 mm i.d., 3 μm, Wilmington, NC, USA) using a Waters HPLC system with a photodiode array detector (Waters, Milford, MA). Operation was conducted under subdued light to avoid carotenoid degradation. Identification of carotenoids was performed by comparison with standard spectra. Quantification was performed using the calibration curve generated with commercially available lycopene, β-carotene, β-cryptoxanthin, lutein, and violaxanthin standards (Sigma-Aldrich).
7
UPLC Analysis of Gingerol Metabolites
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The UPLC analysis was performed on an Acquity UPLC 1-Class system equipped with a binary solvent system, an automatic sample manager, and a photodiode array detector (Waters Corporation). A BEH RP C18 column (2.1 × 50 mm, 1.7 mm) maintained at 40 °C was used for the separation of gingerols (6G,8G, and 10G) and their NADPH-dependent metabolites. The mobile phases consisted of water (A) and acetonitrile (B), both including 0.1% formic acid (v/v), at a flow rate of 0.3 mL min−1. The gradient elution program was optimized as follows: 5% B from 0–0.5 min, 5–10% B from 1–1.5 min, 10–70% B from 1.5–4.5 min.70–95% B from 4.5–4.5 min. 95–95% B from 4.5–5.5 min, 95–5% B from 5.5–6.5 min, 5% B from 6.5–7 min. The injection volume was set at 4 μL and the detection wavelength was set at 280 nm. The phase I metabolites of gingerols were quantitated by UPLC based on the standard curve of the parent compound using the same method as described before.2,19,20 (link) Calibration curves were constructed by plotting each gingerol peak area ratio (Y) versus its concentration (X) using a 1/X2 weighting factor. For 6G (Y6G = 1174.2X + 120.19), 8G (Y8G = 1248.8X − 100.19), and 10G (Y10G = 1010X + 129.88), an acceptable linear correlation was confirmed by correlation coefficients (r2) of 0.9960, 0.9976, and 0.9998, respectively.
8
Quantification of DMBQ in Plant Extracts
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One gram of UWG or EWG was dissolved in 100 mL of deionized water then extracted three times by shaking for 1 min with 300 mL of chloroform. The chloroform layers were obtained and evaporated to dryness using a vacuum evaporator at 35 °C. The dried materials were redissolved in 10 mL of chloroform and filtered through a 0.45 μm polyvinylidene fluoride filter. The standard used for the analysis of DMBQ was purchased from Sigma (St. Louis, MO, USA). DMBQ was analyzed using an HPLC system (Alliance; Waters, Milford, MA, USA) equipped with a photodiode array detector (Waters) operating at 290 nm and a ProntoSIL 120-5-C18 ACE-EPS column (250 × 4.6 mm, 5μm). The injection volume was 5 μL and the flow rate was 0.8 mL/min. Eluent A consisted of 0.1% (v/v) formic acid in deionized water and eluent B was acetonitrile. The solvent compositions for the binary mobile phases were as follows, with each segment lasting 5 minutes: linear gradient 0–25% B, hold at 25% B, linear gradient 25–35% B, hold at 35% B, linear gradient 35–85% B, hold at 85% B, linear gradient 85–0% B, and hold at 0% B.
9
Massetolide Extraction and HPLC Analysis Protocol
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Massetolide extractions and RP-HPLC analysis were conducted according to the methods described previously (de Bruijn et al., 2008 (link); de Bruijn and Raaijmakers, 2009a (link)). Briefly, Pseudomonas strains were grown on Pseudomonas agar plates (Pseudomonas agar 38 g l−1, glycerol 10 g l−1) for 48 h at 25°C. The cells were suspended in sterile de-mineralized water (∼ 40 ml per plate), transferred to 50 ml tubes, shaken vigorously for 2 min and then centrifuged (30 min, 6000 rpm, 4°C). The culture supernatant was transferred to a new tube and acidified to pH 2.0 with 9% HCl. The precipitate was obtained by centrifugation (30 min, 6000 rpm, 4°C) and washed three times with acidified dH2O (pH 2.0). The precipitate was re-suspended in 5 ml dH2O and the pH adjusted to 8.0 with 0.2 M NaOH; the precipitate dissolves. The solution was centrifuged (30 min, 6000 rpm, 4°C) and the supernatant transferred to a new tube and subjected to lyophilization. Analytical HPLC separations were carried out on 5 μm C18 column (Waters Symmetry column, Waters, Etten-Leur, Netherlands), a 55 min linear gradient of 0% to 100% acetonitrile + 0.1% (v/v) trifluoroacetic acid with a flow rate of 0.5 ml min−1. Detection was performed with a photodiode array detector (Waters) at wavelengths from 200 to 450 nm.
10
Analytical Characterization of Compounds
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Ultraviolet spectral were obtained on a Waters 2695 high performance liquid chromatography (HPLC) instrument with a photo diode array detector (Waters, Massachusetts, USA). Preparative HPLC was carried out on a Shimadzu LC-6AD instrument (Shimadzu, Kyoto, Japan) with a UV–Vis detector (SPD-20A), using a YMC-Pack-ODS-A column (250 × 20 mm, 5 μm) (YMC, Kyoto, Japan). The carbohydrates were measured by ion chromatography using an amperometric detector (Metrohm, Herisau, Switzerland) with a Hamilton RCX-30 column (250 × 4.6 mm, 7 μm) (Hamilton, Nevada, USA). Nuclear magnetic resonance (NMR) spectra were obtained using Bruker AV-500 spectrometers (500 MHz for 1H NMR and 125 MHz for 13C NMR) (Bruker, Zurich, Switzerland). Chemical shifts (δ) are given in ppm, with TMS as an internal standard, and coupling constants (J) are in Hz. High-resolution electrospray ionisation-mass spectrometry (HRESIMS) spectra were obtained using an Agilent 6540 ultra-performance liquid chromatography (UPLC) with high-resolution quadrupole time-of-flight (Q-TOF) mass spectrometer (Agilent, California, USA), using an RRHD Eclipse Plus C18 column (150 × 2.1 mm, 1.8 μm) (YMC, Kyoto, Japan)36 (link).
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