The extract of one active strain (Streptomyces sp. IB2014/78-8, S1 Fig ) was cultivated in 1.5 L media of DNPM. The secondary metabolites from the liquid culture were extracted by ethyl acetate as described above. Then, the extract was initially (per minute) separated on an Ultimate 3000 HPLC system (Dionex, Sunnyvale, USA) using a C18 column (Affymetrix, Santa Clara, USA), and a linear gradient of acetonitrile against 0.1% ammonium formate solution in water was flowed over the column at 5 mL/min for 23 minutes. Thus, 23 fractions of separated extract were prepared. The antimicrobial activities of the fractions were analyzed against those of the bacteria described in the paragraph “Biological activity assay of extracts from isolated strains”.
C18 column
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Japan, United Kingdom, Finland
The C18 column is a type of chromatography column used for separating and purifying a wide range of organic compounds. It features a stationary phase consisting of silica particles coated with octadecylsilane (C18) functional groups, which provide a non-polar surface for the retention of non-polar or weakly polar analytes. This column is commonly used in high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC) applications.
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Lab products found in correlation
Ultimate 3000, by Thermo Fisher Scientific (9 mentions)
Q exactive, by Thermo Fisher Scientific (7 mentions)
Easy nlc 1200, by Thermo Fisher Scientific (6 mentions)
Formic acid, by Thermo Fisher Scientific (5 mentions)
High performance liquid chromatography (hplc), by Thermo Fisher Scientific (4 mentions)
Thermo xcalibur 4, by Thermo Fisher Scientific (4 mentions)
Ammonium bicarbonate, by Thermo Fisher Scientific (3 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Q exactive hf x, by Thermo Fisher Scientific (3 mentions)
Lc 20a, by Shimadzu (3 mentions)
Ultimate 3000 hplc system, by Thermo Fisher Scientific (2 mentions)
Ultimate 3000 hplc, by Thermo Fisher Scientific (2 mentions)
Chromeleon computer system, by Thermo Fisher Scientific (2 mentions)
Chromeleon 6, by Thermo Fisher Scientific (2 mentions)
Hplc system, by Thermo Fisher Scientific (2 mentions)
Dionex ultimate 3000 system, by Thermo Fisher Scientific (2 mentions)
Q exactive hf x mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Iodoacetamide, by Merck Group (2 mentions)
Uplc system, by Thermo Fisher Scientific (2 mentions)
Ltq orbitrap velos, by Thermo Fisher Scientific (2 mentions)
178 protocols using c18 column
1
Extraction and Fractionation of Antimicrobial Compounds
2
Metabolic Profiling of Komagataeibacter albida
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K. albida was grown in 20 ml of TSB media for 4 days. 2 ml of pre-culture was inoculated into 50 ml of production media. Six different medias were used: TSB, NL5 (NaCl 1 g/l, KH2PO4 1 g/l, MgSO4x7H2O 0.5 g/l, Trace elements solution 2 ml/l, Glycerol 25 g/l, L-glutamine 5.84 g/l), NL19 (Soy flour 20 g/l, Mannitol 20 g/l), NL111 (Meat extract 20 g/l, Maltose extract 10 g/l, CaCO3 10 g/l), CAS [84 (link)] and SG [99 (link)]. Strain was grown for 7 days at 30°C and 250 rpm. Metabolites were extracted with ethyl acetate from supernatant and acetone-methanol (1:1) mixture from biomass. Extracts were evaporated, dissolved in 100 μl of methanol and samples from biomass and supernatant were combined. 1 μl of each sample were separated on an Ultimate 3000 HPLC (Dionex) using C18 column (Affymetrix) and linear gradient of acetonitrile against 0.1% ammonium formate solution in water. Samples were analyzed on ultrahigh resolution mass spectrometer system maXis (Bruker Daltonics).
3
Ultra-High Resolution Mass Spectrometry Analysis
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The active fractions obtained were analyzed by ultra-high resolution mass spectrometry using an LC-QTOF system maXis II (Bruker, Billerica, USA). The samples were separated on an Ultimate 3000 HPLC system (Dionex, Sunnyvale, USA) using a C18 column (Affymetrix, Santa Clara, USA) and a linear gradient of acetonitrile against 0.1% ammonium formate solution in water at a flow rate 0.5 mL/min for 20 min. The mass detection was performed in both positive and negative modes with the detection range set to 160–2500 m/z. Data were collected and analyzed by Brucker Compass Data Analysis software, version 4.1 (Bruker, Billerica, USA). The screening for known compounds was performed using the Dictionary of Natural Products database version 6.1 (CRC Press, Boca Raton, USA) with the following search parameters: accurate molecular mass, absorption spectra and source of compound isolation [33 ]. Compounds were considered to be similar when the difference in accurate mass was close to 0.01 and the absorption spectra were identical. This part of the study was completed at Helmholtz-Institut für Pharmazeutische Forschung Saarland (HIPS).
4
Quantification of Ellagic and Gallic Acids
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Quantification of EA and GA was conducted on a HPLC (Ultimate 3000, Thermo Fisher Scientific®), coupled to a diode array detector (DAD; Thermo Fisher Scientific®) and equipped with a binary pump (HPG-3x00RS, Thermo Fisher Scientific®), a degasser, and an autosampler equipped with a loop of 20 µL (ACC-3000, Thermo Fisher Scientific®). For data analysis and processing, the software Chromeleon 6.8 (Dionex, Thermo Fisher Scientific®, USA) was used. The wavelength was set at 254 nm for detection of the EA and 270 nm for GA, in accordance with the maximum absorption measured by DAD. The chromatographic separations were achieved with a C18 column (250 mm × 4.6 mm i.d., particle size 5 µm) from Dionex® equipped with a guard column (C18, 4 mm × 3.9 µm, Phenomenex®). Separations were carried out at a column oven temperature of 24°C. The mobile phase consisted of purified water (A) and methanol (B), both acidified with 0.05% trifluoracetic acid (TFA), at a flow rate adjusted to 0.8 mL/min. A gradient program was applied as follows: 0–10 min, 12.5–25% B; 10–15 min, 25–40% B; 15–25 min, 40–75% B; 25–30 min, 75–75% B; 30–33 min, 75–12.5% B.
5
HPLC-DAD Analysis of L. christinae Extract
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L. christinae extract was analyzed by HPLC-DAD. HPLC (Dionex) was composed of an LPG 3X00 pump, an ACC-3000 autosampler, a DAD-3000 (RS) diode array UV/VIS detector, and a column oven. Each sample was injected and isolated through a Dionex C18 column (5 μm, 120 Å, 4.6 mm × 150 mm) at 25°C. The mobile phase consisted of 0.1% TFA water and acetonitrile. The injection volume of samples was 30 μl. The UV wavelength was 205, 254, 280, and 330 nm, respectively, and the chromatograms were acquired at 205 nm.
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L. christinae extract was analyzed by HPLC-DAD. HPLC (Dionex) was composed of an LPG 3X00 pump, an ACC-3000 autosampler, a DAD-3000 (RS) diode array UV/VIS detector, and a column oven. Each sample was injected and isolated through a Dionex C18 column (5 μm, 120 Å, 4.6 mm × 150 mm) at 25°C. The mobile phase consisted of 0.1% TFA water and acetonitrile. The injection volume of samples was 30 μl. The UV wavelength was 205, 254, 280, and 330 nm, respectively, and the chromatograms were acquired at 205 nm.
6
Dual-Loaded PEG-PLA-PEG Aggregates for Drug Delivery
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Dialysis method was used to prepare single-loaded and dual-loaded PEG-PLA-PEG aggregates, using hydrophobic PTX and hydrophilic CDDP as model drugs (Jelonek et al., 2015 , Jelonek et al., 2016 ). Briefly, PEG-PLA-PEG copolymer, PTX, and/or CDDP were dissolved in 1 mL of dimethyl formamide (DMF), a water miscible and low toxic solvent. The solution was added into a pretreated dialysis membrane (MWCO = 3500) and dialyzed in PBS containing 0.1% tween during 24 h to obtain single or dual drug loaded aggregates. Drug release was conducted under constant shaking at 37 °C in 40 mL PBS containing 0.1% tween. The whole release medium was renewed at predetermined time intervals (Li et al., 2018 (link)).
The amount of PTX was determined using high performance liquid chromatography (HPLC; Shimadzu LC-10A) with a Dionex C18 column and UV detector. The mobile phase was acetonitrile/water mixture with 55/45 vol ratio at a flow rate of 1.0 mL/min. Measurements were made at 227 nm. Meanwhile, CDDP was determined by inductively coupled plasma-atomic emission spectroscopy (ICP-AES; Leeman Prodigy XP).
The amount of PTX was determined using high performance liquid chromatography (HPLC; Shimadzu LC-10A) with a Dionex C18 column and UV detector. The mobile phase was acetonitrile/water mixture with 55/45 vol ratio at a flow rate of 1.0 mL/min. Measurements were made at 227 nm. Meanwhile, CDDP was determined by inductively coupled plasma-atomic emission spectroscopy (ICP-AES; Leeman Prodigy XP).
7
HPLC Quantification of Striatal Neurotransmitters
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HPLC with electrochemical detection was used to measure striatal levels of dopamine, DOPAC and serotonin using a method that has been described (Sathe et al., 2012 (link), Nuber et al., 2008 (link)). Briefly, after mice were killed, striata were dissected out and snap frozen directly on dry ice. Striata were then homogenized in 1% perchloric acid (PCA) (1:30 [i.e. tissue weight: PCA volume]) and centrifuged at 14,000g at 4 °C for 20 min. Following centrifugation, 20 μl of the supernatant sample was injected onto a C18 column (Dionex, Germering, Germany). The mobile phase consisted of 90% 50 mM sodium acetate, 35 mM citric acid, 105 mg/L octane sulfonic acid, 48 mg/L sodium EDTA solution, and 10% methanol at pH 4.3. Flow rate was 1 ml/min. Peaks were detected by an ESA Coulochem II electrochemical detector (ESA), and the detector potential was set at 700 mV. Data were collected and processed using the Chromeleon computer system (Dionex).
8
Striatal Neurotransmitter Analysis by HPLC-ECD
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Dissected striatal tissues of mice were prepared for the measurement of DA, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and norepinephrine (NE) with HPLC/ECD analysis. Brain tissue homogenate with 0.1 M HClO4 and 0.1 mM EDTA buffer was centrifuged at 20,000 rpm for 25 min. The supernatant was injected into an autosampler at 4 °C (UltiMate 3000, ESA) and eluted through a C18 column (2.2 μm, 120 Å, 2.1 × 100 mm, DIONEX) with catecholamine analysis mobile phase and was detected by ESA Coulochem III electrochemical detector. The mobile phase consisted of 90 mM NaH2PO4, 50 mM citrate, 1.7 mM 1-octanesulfonic acid, 50 μM EDTA, and 10 % acetonitrile.
9
HPLC-Based Quantification of Striatal Dopamine and DOPAC
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High‐performance liquid chromatography (HPLC) with electrochemical detection was used to measure striatal levels of dopamine, and DOPAC using a method that has been described (Sathe et al., 2012 ). Briefly, mice were killed, 21 days after the last MPTP injection, and the striata were dissected out and snap frozen on solid carbon dioxide. Striata were then homogenised in 0.1 M perchloric acid (1:30 wt/vol), sonicated and centrifuged at 18,600 x g at 4°C for 20 mins. Following centrifugation 20 μl of sample was injected onto a C18 column (Dionex, Germering, Germany) The mobile phase consisted of 90% 50 mM sodium acetate, 35 mM citric acid, 105 mg/L octane sulfonic acid, 48 mg/L sodium EDTA solution, and 10% methanol at pH 4.3. Flow rate was 1 ml/min. Peaks were detected by an ESA Coulochem II electrochemical detector (ESA, Dionex), and the detector potential was set at 700 mV. Data were collected and processed using the Chromeleon computer system (Dionex).
10
Striatal DA and Metabolites Quantification
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The striatal levels of DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were determined by HPLC coupled with electrochemical detection. Briefly, striatal tissues were sonicated in perchloric acid (PCA) (20% wt/vol) containing the internal standard 3, 4-dihydroxybenzylamine (10 mg wet tissue/ml). The homogenate was centrifuged, and an aliquot of the supernatant was injected into HPLC equipped with a C18 column (Dionex, Germering, Germany). The mobile phase was comprised of acetonitrile, tetrahydrofuran, and monochloroacetic acid (pH 3.0) containing of EDTA (50 mg/L) and sodium octyl sulfate (200 mg/L). The amount of DA, DOPAC, and HVA were measured by comparison of peak height ratio of tissue sample with standards and expressed in the quality of wet weight of tissue [12 (link)].
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