Unimax 1010 shaker

Before extraction, the aerial parts of T. diffusa were dried in the shade at room temperature and then crushed into a fine powder. Then, 500 g of the dried selected plant was crushed into a powder and extracted with MeOH at room temperature (3 × 800 mL × 1 h × 200 rpm) with a Heidolph Unimax 1010 shaker. All the extracts were pooled together and evaporated to dryness in vacuo to yield a viscous mass, which weighed 60 g. The chlorophyll contents were eliminated using SPE C18 cartridges, and the samples were eluted with 8 mL of 50%, 70%, and 100% MeOH. Five grams of the fraction obtained using 50% MeOH was subjected to silica vacuum liquid chromatography (silica gel 60 G for thin layer chromatography Merck Millipore, 5 g for sample adsorption, 50 g for the column, Hirsch funnel with pore size M using Cl₂CH₂, AcOEt, AcOEt:MeOH (1:1), and MeOH as eluents (400 mL of each solvent). The fraction eluted with AcOEt (158 mg) was active and was further separated by gravitational chromatography), eluted successively with hexane-AcOEt (3:1, 70 mL) and hexane-AcOEt (2:1, 150 mL). We collected 20 fractions, each containing 10 mL. Fractions 17 and 18 were pooled and evaporated to dryness in vacuo to yield 3.7 mg of yellow oil. The purity of the oil was assessed by TLC, HPLC-DAD, and ¹H-NMR. The proposed structure was established based on the NMR results and comparisons with published results.

For extract preparation, 1 g of smoketree leaves (fresh weight) corresponding to each plant and collection were crushed using a mortar and pestle, then 10 mL of methanol were added, and the samples were vigorously vortexed for 1 min at room temperature (RT). The homogenized samples were placed on a Heidolph Unimax 1010 shaker (Heidolph Instruments GmbH, Germany) and extracted for 2 days under continuous shaking (300 rpm, RT) at room temperature. After this, samples were centrifuged (5 min, 12,000 rpm, 4 °C) using a Universal 320R centrifuge (Hettich, Germany). The supernatant was transferred into a clean tube, the final extract being filtered using a 0.2 µm PTFE filter and stored at −20 °C until needed.

To quantify and isolate the microorganisms from soil samples, a suspension containing an average sample weight of 10 g and 90 mL of sterile tap water was obtained. The suspension was stirred using an Unimax 1010 shaker (Heidolph Instruments, Schwabach, Germany) at 120 rpm for 10 min, and then left for 5 min to precipitate the solid phase. The obtained soil suspensions and seawater samples were used for inoculating the liquid selective nutrient media by the method of serial ten-fold dilutions. The results were evaluated by the most probable number method according to the McCredy table [27 ].