Rotary evaporator

The herbs were washed thoroughly under running tap water and dried under shade for three weeks. Dried spices and herbs were subjected to a fine powder and stored at room temperature in air-tight containers.Then, 200 g fine powder of each plant was macerated for five days, using analytical grade ethanol (1000 mL), in air-tight glass bottles. Maceration was performed to increase the contact between plant material and solvent (Ethanol) and to soften plant’s cell wall so that plant phytochemicals soluble in ethanol may be released. Sonication (after maceration) was added to further enhance the disruption of the plant cell wall and facilitate the release of phytochemicals. The extracts were filtered using Whatman #1 filter paper (Sigma, USA). Extracts were concentrated by vacuum evaporation in a rotary evaporator (Buchi, Switzerland) and dried in a vacuum hood at 40 °C, and stored at 15 °C till further use. The extracts were filtered using Whatman #1 filter paper. Extracts were concentrated by vacuum evaporation in a rotary evaporator (Buchi, Switzerland) and dried in a vacuum hood at 40 °C, and stored at 15 °C till further use.
The extracts, once dried, were weighed to find out % recovery by the following formula: $$\mathrm{Extract}\mathrm{recovery}\left(\%\frac{w}{w}\right)=\frac{\mathrm{A}}{200}\times 100$$
where A = weight of dry extract.

The root part of the plant was ground to the fine powder using laboratory grinder. For obtaining of phenolic-enriched extract, 100 g of dried root powder was added into the 2L round bottom flask and 1000 mL of methanol (80% v/v), and 50 mL of hydrochloric acid (6 M) were added. The mixture was refluxed (90 °C, 2 h) [20 ]. The obtained extract was filtered and the solvent was evaporated (60 °C) by using a rotary evaporator (Buchi, Flawil, Switzerland) [21 (link)]. To obtain the phenolic-enriched extract (PRE) the dried extract was re-extracted with different polarity solvents including hexane, chloroform, ethyl acetate, n-butanol, and water, respectively. For extract partitioning, 250 mL from each solvent was used and re-extraction was performed twice in a separating funnel. Finally, the obtained extract was filtered and concentrated using a rotary evaporator (Buchi. Switzerland). The total phenolic content of each extract was determined colorimetrically using a visible spectrophotometer (Novaspec II Visiblespectro, Japan) at 765 by Folin-ciocalteu assay [21 (link)], and results were reported as mg gallic acid equivalent (GA eq.) /g dry crude extract. The extract with the highest concentration of phenolic compounds is called phenolic-enriched extract (PRE) and is used for further experiments.

Shrimp shells were scraped free of meat, rinsed with running tap water, and air dried at room temperature. The shells were blended with food grade ethanol in the ratio of 1:2 (w/v). The mixture was filtered and further evaporated to remove ethanol by using Büchi rotary evaporator (Büchi Labortechnik AG, Flawil, Switzerland) at 40 °C. The RP-HPLC was performed to measure AST content in the extract. The analyses were carried out in isocratic conditions by using BDS hypersil C18 column (150 mm × 4.6 mm) packed with 5-mm diameter particles. The mobile phase was methanol/acetonitrile (50:50), the flow rate was 1 mL/min, and the detection wavelength was set at 480 nm. The chromatographic peaks of AST in the extract were identified by comparing the peaks of AST standard and expressed AST content as mg/g extract.

OA was isolated from Syzygium aromaticum [(Linnaeus) Merrill & Perry] (Myrtaceae) (cloves) using a protocol previously validated in our laboratory with minor modifications [7 (link),10 (link),20 (link)]. Briefly, air-dried S. aromaticum flower buds (500 g) were exhaustively extracted sequentially with dichloromethane (DCM) and ethyl acetate (EA) (twice with 1 L for 24 h for each solvent) at room temperature. The plant material was removed by filtration and the resultant filtrates were concentrated in vacuo at 60 ± 1°C using a Büchi rotary evaporator (Buchi Labortechnik AG, Flawil, Switzerland) to obtain DCM soluble (63 g) and ethyl acetate soluble (EAS, 85 g) crude extracts. Crude EAS have been previously shown to contain OA, ursolic acid, methyl maslinate and methyl corosolate [21 (link)]. Hence subjecting EAS to column chromatography and recrystallisation from methanol yielded pure OA whose structure was confirmed by spectroscopic analysis using ¹H and ¹³C nuclear magnetic resonance (NMR). Spectroscopic data indicated that the S. aromaticum-isolated OA (compound 1 in Fig 1) was pure and similar to commercial OA, hence this triterpene was used for animal studies and as a starting material for the synthesis of oleanane derivatives.

Melting temperatures were determined on an electrothermal melting point apparatus (England) and are uncorrected. Thin-layer chromatography (TLC) was performed on Kieselgel GF_254, and spots were detected by spraying with 1% H₂SO₄, followed by heating at 150–200 °C. Column chromatography was performed with silica gel G₆₀. ¹H-NMR (400 MHz) and ¹³C-NMR (100 MHz) spectra were recorded for solutions in CDCl₃ with TMS as an internal standard at the WMSRC Jahangirnagar University, Savar, Dhaka, Bangladesh. Infrared spectral analyses were recorded using a Fourier-transform infrared (FTIR) spectrophotometer (IR Prestige-21, Shimadzu, Japan) within 200–4000 cm⁻¹ at the Department of Chemistry, University of Chittagong, Bangladesh. Mass spectra of the synthesized compounds were obtained by liquid chromatography-electrospray ionization tandem mass spectrometry in positive ionization mode. All evaporations were conducted under reduced pressure using Büchi rotary evaporator (Germany).