Alugram xtra sil g uv254

If not otherwise stated, the different chemicals for the synthesis of CBV4-57 were obtained from Sigma Aldrich (Sigma Aldrich S.a.r.l; Saint-Quentin-Fallavier, France). Furthermore, unless otherwise noted, all experiments were carried out under a nitrogen atmosphere. Solvents (CH₂Cl₂ and THF) were dried via a purification solvent system MB-SP- 800 (MBRAUN, Garching, Germany). Melting points (mp) were obtained on a on a Mettler-Toledo MP50 apparatus (Mettler-Toledo, Columbus, OH, USA)and are uncorrected. IR spectra were recorded on a Thermo Nicolet Nexus spectrometer (Thermofisher Scientific, Waltham, MA, USA). NMR spectra were recorded on Bruker Avance 300 MHz spectrometers (Bruker, Wissembourg, France). The NMR spectra were acquired in CDCl₃, and the chemical shifts were reported in parts per million referring to CHCl₃ (δ_H 7.26 for proton and δ_C 77.16 for carbon). Signals are described as follows: s, singlet; brs, broad signal; d, doublet; t, triplet; m, multiplet. HRMS data were recorded on a Xevo G2 QTOF instrument (Waters, Milford, MA, USA). Reactions were monitored by TLC on silica gel Alugram® Xtra SIL G/UV₂₅₄ (Macherey-Nagel, Duren, Germany). Column chromatography was performed on Machery-Nagel silica gel 0.063-0.2 mm.

Thin-layer chromatographic (TLC) plates, composed of a 0.2 mm thick precoated silica gel 60F-254 layer (precoated TLC-sheets ALUGRAM Xtra SIL, G/UV 254 from Macherey-Nagel, Germany), received 1 μL of the propolis extracts (containing 50–100 μg of propolis extract), placed 1 cm from the lower edge of the plate. Preliminary TLC separation of the extracts was performed using different solvent systems; the solvent front was allowed to travel to at least 75% of the TLC plate height. Two solvent systems, n-hexane : ethyl acetate : formic acid (30 : 70 : 1) as a combination of nonpolar solvents and ethyl acetate : methanol : formic acid (50 : 50 : 1) as a combination of polar solvents, were used to elute the compounds in the extracts [18 (link)]. The spots were visualized using natural light, short wave ultraviolet (254 nm), and long wave ultraviolet (366 nm) for detecting the fluorescence compounds and by spraying with an anisaldehyde-sulfuric acid agent followed by heating at 110°C for detecting triterpenoids [19 (link)]. Finally, the TLC plates were photographed by a TLC visualizer 2 (CAMAG, Muttenz, Switzerland). Each spot was marked, and the retardation factor (Rf) value was calculated. The Rf value of the separated compound was defined as the distance travelled by the compound divided by the distance travelled by the solvent (the solvent front).

Fractionation of active constituents in the crude leaf extract was performed on silica-packed column chromatography. Silica (200 g; Kiesegel 60 M [0.004–0.063 mm mesh size]; Macherey-Nagel GmbH & Co.KG, Düren, Germany) was packed in 40 × 330 mm column and conditioned with analytical grade n-hexane (Sigma Aldrich, St. Louis, USA) for 3 h prior sample loading. Thirty-five grams of the leaf extract were loaded onto the packed silica and elution of various fractions achieved through gradient mobile phase of analytical grade n-hexane and ethyl acetate (100:0–0:100) and finally methanol (Sigma Aldrich, St. Louis, USA). Fractions were chromatographed on thin layer chromatography (TLC) silica plates (ALUGRAM® Xtra SIL G/UV₂₅₄ [0.2 mm], Macherey-Nagel GmbH & Co.KG, Düren, Germany) developed with n-hexane and ethyl acetate (1:2 v/v) as mobile phase. The plates were air-dried, sprayed with 30 % sulfuric acid and baked in an oven for detection under UV lamp (λ_{254–365 nm}). Based on TLC monitoring and evaluations, fractions with similar retention factor (Rf) values were pooled together, rotor-evaporated and assayed for activity against mosquito larvae.

TLC plates of 5 × 10 cm size with fluorescence indicator (TLC Silica gel 60 F₂₅₄; Merck KGaA, Darmstadt, Germany, or ALUGRAM^® Xtra SIL G UV254, Macherey–Nagel GmbH & Co. KG, Düren, Germany) were used. Sample and reference solutions were applied to these plates manually, using 2 µl capillaries (minicaps^®, Hirschmann Laborgeräte GmbH & Co. KG, Eberstadt, Germany). Two different spotting patterns were evaluated, shown in Supplementary Fig. S1. Plates were developed as described in the respective monographs of the GPHF Minilab Manual⁴⁹ . The composition of the mobile phases is given in Supplementary Table S1. After development, the plates were left to dry completely for at least 30 min at room temperature. The spots of the APIs were detected under UV light using a battery-operated hand lamp (MINI-UV Test Lamp, 256 nm, Prinz Verlag GmbH, Passau, Germany). Storage of the developed plates for up to three days, protected from light and humidity, did not affect the quantitative evaluation for the investigated APIs. No chemical staining of the spots was carried out in this study.

Instrumentation included JEOL spectrometer with tetramethylsilane as an internal standard for ¹H (500 MHz) and ¹³C (125 MHz) NMR spectra. Materials used in chromatography; normal-phase silica gel for column chromatography (Fluka^®, St. Louis, Mo, USA, 230–400 mesh), pre-coated TLC-plates ALUGRAM Xtra SIL G/UV254 (MACHEREY-NAGEL^®, Düren, Germany, 0.2 mm) (normal phase), Sil G-25 unmodified standarad silica layers on glass for Preparative TLC, layer thickness 2 mm (MACHEREY-NAGEL^®, Düren, Germany) and Sephadex LH-20 (Sigma Aldrich^®, Darmstadt, Germany). Anisaldehyde–sulfuric acid was used as a spraying reagent.

A standard working solution of citral was prepared by diluting aliquots of >98% stock solutions in methanol. Overnight bacterial cultures were diluted into 25 mL of fresh MHB and incubated with shaking (250 rpm) at 37°C. When cultures reached an absorbance of A_600nm = 0.8, citral was added to a 2 or 4 mg/mL final concentration. Aliquots were removed at t_0h, t_1h, t_3h, and t_24h post-exposure; the growth medium was then collected by centrifugation and filtration through two filters of 0.45 and 0.2 μm pore size, respectively. An organic extraction was repeated three times using 1 mL dichloromethane (for a total volume of 3 mL). The organic fractions were pooled and dried overnight in a chemical hood, and were finally re-dissolved in 500 μL of dichloromethane. Organic fractions and standards were diluted (1:200 for organic fraction and 1:2,000 for standard) in methanol. Ten microliter volumes were then sprayed as 8 mm bands on a TLC plate (Alugram^® Xtra SIL G UV254, Macherey-Nagel) using an automatic sampler (ATS4, Camag, Moirans, France) connected to visionCATS Camag TLC software V2.4. The TLC plates were developed in an automatic developing chamber (ADC 2, Camag) with a mobile phase containing cyclohexane:ethyl acetate (7:2) over a 70 mm migration distance. Spots were observed using UV-light at 254 nm (CV-415.LS, Uvitech, England).

Total lipids were extracted using the method of Bligh and Dyer, as modified by Ames [26 (link),27 (link)]. E. coli cells were induced in 25 mL of culture, as previously described for protein expression except that induction was reduced to 4 hours. Cells were then harvested with a 4 000 x g centrifugation for 10 min, the pellet was re-suspended in 0.8 mL of PBS, and 2 mL of methanol and 1 mL of chloroform were added with thorough mixing. After 1 h at room temperature, 1 mL of chloroform and 1 mL of PBS solution were added and then the phases were thoroughly mixed. After a brief low-speed centrifugation, the resulting organic phase was carefully removed, dried under N₂, re-suspended in a small volume of chloroform and then analyzed for individual phospholipids. Two-dimensional thin layer chromatography, on pre-coated TLC-sheets ALUGRAM^® Xtra SIL G/UV₂₅₄ (Macherey Nagel), was used to separate the different lipids. For the first dimension, a solution of chloroform / methanol / water (65:25:4, v/v/v) was used and then a chloroform / methanol / acetic acid (65:25:10, v/v/v) solution was employed for the second dimension. After drying the plates, lipids were visualized by exposure to iodine vapor.