All the reagents and chemicals were of high purity and analytical grade. Standard paclitaxel was procured from MP Biomedicals (USA). Media components used for the growth and maintenance of taxol-producing endophytes were purchased from Hi-Media (Mumbai). The electrophoresis reagents, molecular grade chemicals used for DNA isolation were purchased from SDFCL (India) and Sigma-Aldrich (USA), respectively. Pre-coated Silica gel 60, F254 TLC plates, and HPLC solvents were of HPLC grade and procured from Merck (Germany). Universal primers for ITS and dbat genes were procured from Sigma (USA) and Bioservice, respectively.
F254 tlc plate
Manufactured by Merck Group
Sourced in Germany
F254 TLC plates are thin-layer chromatography (TLC) plates that contain a fluorescent indicator. These plates are designed for the separation and analysis of compounds using TLC techniques. The F254 designation indicates the presence of a fluorescent substance within the adsorbent layer, which can be used to visualize separated compounds under ultraviolet (UV) light.
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Lab products found in correlation
Linomat 5, by CAMAG (2 mentions)
Wincats software, by CAMAG (2 mentions)
Tlc scanner 3, by CAMAG (2 mentions)
Pre coated silica gel 60, by Merck Group (1 mentions)
Hplc solvents, by Merck Group (1 mentions)
Avance 400 mhz spectrometer, by Bruker (1 mentions)
Tlc plate heater, by CAMAG (1 mentions)
Automatic tlc sampler 4 ats4, by CAMAG (1 mentions)
Tlc visualizer 2, by CAMAG (1 mentions)
Betulinic acid, by ChemFaces (1 mentions)
Lupeol, by ChemFaces (1 mentions)
Tlc visualizer, by CAMAG (1 mentions)
Sb c18 column, by Agilent Technologies (1 mentions)
Avance 3 600 mhz spectrometer, by Bruker (1 mentions)
Twin trough glass chamber, by CAMAG (1 mentions)
Linomat 5 automatic sample spotter, by CAMAG (1 mentions)
Standard iaa, by Merck Group (1 mentions)
Linomat 4 autosampler, by CAMAG (1 mentions)
Tlc scanner 4, by CAMAG (1 mentions)
Tina software, by Elysia raytest (1 mentions)
15 protocols using f254 tlc plate
1
Paclitaxel production by endophytes
2
Rapid DPPH Antioxidant Screening
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DPPH (2,2-Diphenyl-1-picrylhydrazyl) assay [13 (link)] was used as a rapid thin layer chromatography screening method to evaluate the antioxidant activity of the methanolic extracts due to free radical scavenging. DPPH is a purple coloured stable free radical, which on reduction gives yellow coloured diphenyl picryl hydrazine compound. 2.5 μL of different methanolic extracts (1 mg/mL) were loaded on silica gel F254 TLC plates (Merck, Germany) which were sprayed with 0.05% DPPH solution in methanol and examined at 30 minutes after spraying. Any antioxidant compound is seen as a yellow spot on a purple background. Vitamin C and gallic acid were used as positive controls.
3
Isolation and Characterization of Compounds from H. petiolare EO
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A silica slurry of 3.547 g of H. petiolare EO was packed in a silica gel column (40 cm × 4 cm). The separation was performed using a gradient elution of hexane: ethyl acetate (Hex: EA) in order of increasing polarity from 100:0 to 94:6 (Hex: EA). The separation yielded 53 fractions (20–50 mL) labeled as 1–53 which were further concentrated at 45 °C.
Fraction 29 was subjected to preparative thin-layer chromatography (prep TLC) to purify compound1 . Fraction 29 (20 mg) was dissolved in 750 μL of hexane then 250 μL was loaded on three individual silica gel 60 F254 TLC plates (20 cm × 10 cm; Merck, Germany). Subsequently, the plates were developed at 97:3 hexane: ethyl acetate (double run). Compound 1 was marked under λ254 nm, scrapped off, and eluted with hexane. Compound 2 was purified from fraction 31 by prep TLC at 92:8 hexane: ethyl acetate (double run) as previously described.
The MS spectra of compound1 and compound 2 were obtained by dissolving 0.5 mg in 300 μL of hexane and analyzing the samples by the method previously described. Their 1H NMR and 13C NMR spectra were recorded at 20 °C using deuterated chloroform on a Bruker Avance™ 400 MHz spectrometer (Germany). The chemical shifts of 1H and 13C in ppm (δ) were determined with tetramethylsilane (TMS) used as an internal reference.
Fraction 29 was subjected to preparative thin-layer chromatography (prep TLC) to purify compound
The MS spectra of compound
4
TLC Analysis of Herbal Extracts
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For each of the herbal samples, the test solution was prepared by extracting 2 g dried and pulverized herb with 20 ml methanol under ultrasonic condition at room temperature (approximately 21 °C) for 60 min, followed by filtration. The filtrate was then evaporated to dryness under reduced pressure at 50 °C. The extract was dissolved in 5 ml of methanol and was used for TLC analysis on silica gel 60 F254 TLC plates (20 cm × 10 cm, Merck, Germany). Extracts (2 μL) were applied to the plates as 8 mm bands using the CAMAG automatic TLC Sampler 4 (ATS4, Muttenz, Switzerland), development to a distance of 8.5 cm up the plate was performed in a TLC developing chamber. A mixture of ethyl acetate: methanol: water (8:1:1, v/v, upper layer) was used as the developing solvent system. The plate was then heated on a TLC plate heater (CAMAG, Muttenz, Switzerland) at about 105 °C after spraying with the 10 % solution of sulfuric acid in ethanol until the color of the spots appeared distinctly. High-definition images of the TLC plate were captured using a Visualizer 3 (CAMAG, Muttenz, Switzerland) linked with WinCATS software28 (link) under UV light (λ = 366 nm).
5
Thin Layer Chromatography Analysis of Triterpenoids
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TLC analysis was carried out using activated silica gel 60 F254 TLC plates (Merck, Germany) as the stationary phase. Betulinic acid (Chemfaces, China) and lupeol (Chemfaces, China) were utilized as standard references. Five microliters of each 1 mg/mL standard reference solution and each 15 mg/mL sample extract were loaded with a 4 mm bandwidth on TLC plates using a sample applicator (Linomat V, Camag, Switzerland) equipped with a 100 μL syringe. Then the plates were developed in the optimized mobile phase to a distance of 80 mm. After that, the dried plates were sprayed with anisaldehyde-sulfuric acid reagent before drying in an oven at 105°C for 20 min. The TLC chromatograms were viewed and photo-recorded under UV light at 366 nm using a TLC viewer (TLC visualizer 2, Camag, Switzerland). The results from this method were orderly rearranged using visionCATS version 2.3 software.
6
HPTLC Analysis of Nitidine Extract
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High-performance thin layer chromatography analysis of the extract was carried out on Camag HPTLC system consisting of Linomat V semi-automatic applicator, Camag syringe of 100 μL capacity, Camag twin trough chamber (20 × 20 cm), Camag TLC scanner 3, equipped with win CATS software (version 1.4.3, Camag, Muttenz, Switzerland). Aluminum backed silica gel 60 F254 TLC plates (10 × 10 cm, 0.2 mm thickness, E-Merck) were used for separation and identification of nitidine. The mobile phase was chloroform: Methanol (7:1, v/v). Densitometric scanning at 332 nm was performed.[8 ]
7
HPTLC Analysis of Prunus domestica Extracts
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For HPTLC analysis, CAMAG Linomat V (CAMAG) applicator with precoated silica gel F254 TLC-plates (Merck, Darmstadt, Germany) was used. The extracts of P. domestica fruit in different solvents (chloroform, methanol, water:alcohol (1:1; v/v) and water) were concentrated using a rotary evaporator and dried under reduced pressure using N2 gas inert condition. Finally, 5 mg/mL stock solution of each extract was prepared and spotted on TLC plates. For analysis, properly diluted stock solution was spotted via TLC plates, followed by the development of TLC plates in toluene:ethyl acetate:formic acid (5:4:0.5; v/v/v) solvent system for chloroform, methanol, aqueous:alcohol (1:1; v/v) extracts, whereas butanol:acetic acid:water (8:2:2; v/v/v) solvent system was used for aqueous extract TLC plate development. TLC plates were scanned by CAMAG scanner-3 at 366 nm, and photographs of the chromatograms were saved [15 (link)].
8
Characterization of Forced Degradation Products
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NMR spectra were obtained using a Bruker Avance-III 600 MHz spectrometer (Bruker Corp, Karlsruhe, Germany) in CD3OD with tetramethylsilane (TMS) as the internal standard. The liquid chromatography-mass spectrometry (LC-MS) system (Agilent 6410 Triple Quad, CA, USA; Q-TOF, Waters, USA) was used to analyse the forced degradation products. LC was performed on an Agilent SB-C18 column (1 μL/mL, 4.6 mm × 250 mm, 5 μm) at 30°C, and the mobile phase consisted of a mixture of methanol and water (70:30, v/v). Mass spectrometry analyses were performed in the positive ionization mode with a scanning range of m/z 80 ~ 1, 000. The following electrospray ionization (ESI) parameters were set: the capillary voltage was 4,000 V with a target mass-to-charge ratio of 150, the nebulizer (N2) pressure was set to 30 psi, the drying gas (N2) temperature was 325°C, and the drying gas flow rate was 7 L/min. For the MS/MS investigations, the ions of interest were isolated through quadrupole tandem mass spectrometry. Thin-layer chromatography (TLC) was performed on silica gel 60 F254 TLC plates (Merck, Darmstadt, Germany) and developed using a cyclohexane:acetone mixture (5: 1, v/v). After drying, the TLC plates were examined under UV light at 254 nm in a TLC visualizer (Camag, Muttenz, Switzerland).
9
TLC Fingerprinting of B. suffruticosa Extract
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n-Hexane extract (20 mg) of B. suffruticosa was dissolved in 40 ml of n-hexane, treated with a pinch of charcoal, filtered and the volume was made up to 50 ml in a volumetric flask. This sample solution was used for the thin layer chromatography (TLC) fingerprinting profile. TLC plates consisted of 10 cm × 10 cm, precoated with silica gel 60 F254 TLC plates (E. Merck) (0.2 mm thickness) with aluminum sheet support. The spotting device was a Camag Linomat V Automatic Sample Spotter (Camag, Muttenz, Switzerland); the syringe, 100 µl (Hamilton). The developing chamber was a Camag glass twin trough chamber (20 cm × 10 cm); densitometer a Camag TLC Scanner 3 linked to winCATS software (Camag, Muttenz, Switzerland). The experimental conditions were kept constant where, the temperature was 25°C ± 2°C and relative humidity was 40%. TLC fingerprint was developed by applying 25 µl of n-hexane extract (100 mg/50 ml) in a duplicate along with standards, lupeol and β-sitosterol with band size of 8 mm, and distance between the tracks of 12 mm. Plate was developed in a solvent system of toluene: Methanol (9.3:0.7), dried and observed under ultraviolet (UV) 254 nm and UV 366 nm. The plate was derivatized with anisaldehyde-sulfuric acid reagent followed by heating at 100°C until the colored band appeared. The RF value and color of the resolved bands were noted.
10
Characterization of Organic Compounds
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All commercially available reagents and solvents were purchased from Alfa Aesar (Ward Hill, Massachusetts, MA, USA) and used without any further purification. Melting points were determined on Büchi apparatus and were uncorrected. 1D (1H NMR, 13C NMR) and 2D (COSY, NOESY, HMBC, HSQC-DEPT135) spectra were carried out on 400 or 600 MHz Bruker spectrometers, respectively Avance™ DRX and III instruments (Bruker BioSpin GmbH, Rheinstetten, Germany). Chemical shifts (δ) are expressed in ppm while coupling constants (J) in Hz. The multiplicity of vertices is expressed as s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), and m (multiple). Flash chromatography was performed on Merck silica gel (40–63 μm) with the indicated solvent system using gradients of increasing polarity in most cases (Merck KGaA, Darmstadt, Germany). The reactions were monitored by analytical thin-layer chromatography on pre-coated silica gel 60 F254 TLC plates, 0.25 mm layer thickness (Merck KGaA, Darmstadt, Germany). Mass spectra were recorded on a UPLC Triple TOF-MS: Acquity UPLC (Waters, Milford, MA, USA), Triple TOF-MS 5600+ (AB Sciex LLC, Framingham, MA, USA).
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