Rotary evaporator

The solvent fractions were obtained using chloroform, n-butanol, and distilled water in increasing order of their polarity. The fractionation was done according to the method described by Mengie et al. [39 (link)]. Eighty gram (80 g) of 80% methanol crude extract of W. somnifera was dissolved in 400 ml of distilled water in a separator funnel. An equal volume of chloroform was added to the aqueous solution, and they were mixed well by gentle agitation. Then, the mixture was allowed to form a distinct layer (the chloroform at the bottom and the aqueous solution at the top). After 24 h, the chloroform fraction was isolated from the mixture, and the process was repeated twice with fresh chloroform. All the chloroform fractions were collected together and subjected to evaporation via a Rotary Evaporator (Yamato, Japan), which was set at 40°C to obtain the chloroform fraction. The remaining aqueous residue was further fractionated with 400 ml of n-butanol in the same manner as chloroform. The remaining aqueous residue after removal of n-butanol was frozen in a deep freezer overnight. Then, the frozen residue was dried with a lyophilizer (Operon, Korea Vacuum Limited, Korea). The three fractions were labeled and kept in the deep freezer with airtight containers in the refrigerator at −20°C until they were used in the antidiarrheal test.

Briefly, CBLs were prepared via two steps. First, Lec/Chol/CHR, dl-α-tocopherol, and octadecyl amine (80:20:1, mass ratio) were dissolved in anhydrous ethanol and then evaporated using a rotary evaporator (Yamato, Japan) at 60 °C under reduced pressure to produce the thin film. Then, the sample was dried at room temperature for 2 h and then hydrated with 10 mL of sodium citrate buffer (pH = 3.47) at 40 °C to induce phase transition. The CHR liposome suspensions were sonicated at 200 W for 3.3 min and filtered using a 0.22 μm microporous membrane filter to achieve the desired size. Then, the sorted CHR liposomes were added to a 0.4 mg/mL BBH solution at a volume mass ratio of 1:1 (Lec/BBH = 25:1, mass ratio), and then the pH of the mixture was adjusted to 7.0, followed by incubation in a water bath at 60 °C for 20 min to produce the CHR-BBH compound liposomes. The drug concentration of BBH was 0.1600 mg/mL and 0.0500 mg/mL of CHR in CBLs.

The crude methanolic extract was obtained from 200 mg of sample (fine flour) suspended in 8 mL of methanol (ACS, Tedia, CRT-Mexico). The mixture was placed into 50 mL centrifuge tubes and sonicated for 30 min (FS20, Fisher Scientific, Waltham, MA, USA), during which they were mixed and vortexed (VX100, Labnet, Madrid, Spain) every 10 min. Then, the samples were incubated (VWR Avantor, Radnor, PA, USA) for 15 h (25 °C/darkness, 200 rpm) and centrifuged at 4000 rpm/10 min in a Hermle Z 366 centrifuge (Labortechnik GmbH, Wehingen, Germany). The supernatant was decanted and stored at −20 °C, and the pellet was re-extracted for 2 h and processed again as described before. The supernatants from the two extractions were combined and concentrated to dryness in a rotary evaporator under reduced pressure (Yamato, Santa Clara, CA, USA) at 37 °C and reconstituted in 1 mL of methanol (ACS, Tedia, CTR-Mexico). The antioxidant capacity of the methanolic extracts was determined by the DPPH and ORAC methods as described by Cardador-Martínez, et al. [22 (link)] and Prior et al. [23 (link)], respectively. The results were expressed as µmol equivalents of Trolox/g of flour.

F. thonningii stem barks were washed with distilled water to remove dirt and dust. The cleaned plant materials were dried at room temperature. The plant materials were grounded into coarse powder by the electrical mill. Then, the coarse powdered plant materials were macerated separately in hydro-methanol solvent for about 72 hours, then the plant materials were filtered via Whatman No. 1 filter paper and remacerated three times with fresh hydro-methanol solvent. The filtrates of each successive maceration were concentrated by using a Rotary evaporator (Yamato Rotary evaporator, Japan) adjusted to a temperature of 40°C. Finally, the semidried residues were frozen in the deep freezer and dried using a lyophilizer (Labfreez, China) to entirely remove the remaining solvent [15 , 23 ].

The CFS of MSI45 was mixed with an equal volume of six different solvents such as chloroform, ethyl acetate, ethanol, dimethyl ether, dimethyl sulfoxide and methanol in separate extraction flasks and kept for overnight incubation at 4 °C. The solvents were then evaporated in a rotary evaporator (Yamato) and the remaining residues were concentrated in vacuum concentrator (Labconco) and lyophilised in a lyophilizer (Yamato). The lyophilised compounds were dissolved at a concentration of 50 mg L⁻¹ and from this 100 μl of the compound MSI45 was tested for antimicrobial activity using a well diffusion assay.