The broccoli seeds were soaked in water and placed in the dark for 8 h. The seeds were then poured on the sieve and placed in the dark for 7 days. The broccoli seeds were washed with purified water every 12 h. For the preparation of the BSE, the broccoli sprouts were dried in an oven at 40°C (Memmert, Germany) and pulverized. The broccoli sprout powder (0.1 g) was then added to 80% v/v methanol (20 ml) and stirred for 24 h. After that, the filtration was performed using Whatman paper No. 1. The solvent was then evaporated by a rotatory evaporator (Heidolph, Germany) at a temperature of 45°C.
Rotatory evaporator
Manufactured by Heidolph
Sourced in Germany
A rotatory evaporator is a laboratory instrument used to remove solvents from samples through controlled evaporation. It consists of a rotating evaporation flask, which is submerged in a heated liquid bath, and a condenser system that collects the distilled solvent.
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7 protocols using rotatory evaporator
1
Broccoli Sprout Extract Preparation
2
Assessing β-Carotene Bleaching Inhibition by EAP
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The ability of EAP to prevent bleaching of β-carotene was determined as described by Koubaa et al. [55 (link)]. A stock solution of β-carotene/linoleic acid mixture was prepared by dissolving 0.5 mg of β-carotene, 25 µL of linoleic acid, and 200 µL of Tween 40 in 1 mL of chloroform. After evaporation of the chloroform under vacuum in a rotatory evaporator (Heidolph Instruments GmbH & Co. KG, Schwabach, Germany) at 40 °C, 100 mL of bi-distilled water was added, and the resulting mixture was vigorously stirred. The emulsion obtained was freshly prepared before each experiment. Aliquots (2.5 mL) of the β-carotene/linoleic acid emulsion were transferred to test tubes containing 0.5 mL of each EAP at different concentrations (1–5 mg/mL). After incubation for 2 h at 50 °C, the absorbance of each sample was measured at 470 nm. BHA was used as a positive standard. A control consisting of 0.5 mL distilled water was used instead of the sample. The antioxidant activities of EAP were evaluated in terms of β-carotene bleaching according to Equation (6).
where A0 and A2 are the absorbance values measured in the presence of EAP at t = 0 and t = 2 h, respectively. A00 and A02 are the absorbance values measured in the absence of EAP at t = 0 and t = 2 h, respectively.
where A0 and A2 are the absorbance values measured in the presence of EAP at t = 0 and t = 2 h, respectively. A00 and A02 are the absorbance values measured in the absence of EAP at t = 0 and t = 2 h, respectively.
3
Ethanol Pretreatment and Aqueous Extraction
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The sample powder was pretreated with 70% ethanol twice to remove small molecular compounds. Then, the deionized water was added to the sample powder in a ratio of 5 : 1, and the mixture was kept boiling for 2 hours. Finally, the extract was filtered and concentrated in a dry solid form using a rotatory evaporator (Heidolph, Germany).
4
Extraction and Characterization of Balanites aegyptiaca and Petroselinum sativum
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Balanites aegyptiaca fruits and Petroselinum sativum leaves were purchased from local markets in Assiut Governorate. The pericarp of Balanites aegyptiaca fruits was cleaned and then prepared in the form of coarse powder. Petroselinum sativum leaves were washed with tap water, dried in the shade for one week, and stored in well-sealed cellophane bags. The dried leaves were powdered to be used for extract preparation. According to Gad and coworkers [6 (link), 12 ], one kilogram of the dried powdered plant materials was extracted with four liters of boiling distilled water using percolation for 48 hours. The extracts were filtrated. Then, the filtrate was concentrated under reduced pressure by rotatory evaporation using a rotatory evaporator (Heidolph, Germany) at 40°C until the therapeutic residues were obtained. The therapeutic liquids were subjected to lyophilization using the freeze dryer (VirTis, USA) until fine powder materials were obtained and weighed to give 200 grams of dried powdered extract in the case of Balanites aegyptiaca and 150 grams in the case of Petroselinum sativum. Thus, the yielded extract was about 20% w/w and 15% w/w for Balanites aegyptiaca and Petroselinum sativum, respectively. The extracts were dissolved in distilled water before administration to diabetic and normal rats. The extracts were administered orally using an orogastric tube.
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Balanites aegyptiaca fruits and Petroselinum sativum leaves were purchased from local markets in Assiut Governorate. The pericarp of Balanites aegyptiaca fruits was cleaned and then prepared in the form of coarse powder. Petroselinum sativum leaves were washed with tap water, dried in the shade for one week, and stored in well-sealed cellophane bags. The dried leaves were powdered to be used for extract preparation. According to Gad and coworkers [6 (link), 12 ], one kilogram of the dried powdered plant materials was extracted with four liters of boiling distilled water using percolation for 48 hours. The extracts were filtrated. Then, the filtrate was concentrated under reduced pressure by rotatory evaporation using a rotatory evaporator (Heidolph, Germany) at 40°C until the therapeutic residues were obtained. The therapeutic liquids were subjected to lyophilization using the freeze dryer (VirTis, USA) until fine powder materials were obtained and weighed to give 200 grams of dried powdered extract in the case of Balanites aegyptiaca and 150 grams in the case of Petroselinum sativum. Thus, the yielded extract was about 20% w/w and 15% w/w for Balanites aegyptiaca and Petroselinum sativum, respectively. The extracts were dissolved in distilled water before administration to diabetic and normal rats. The extracts were administered orally using an orogastric tube.
5
Synthesis of Stable PET Nanoparticles
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PET NPs was prepared based on the previous method with slight modifications70 (link). Briefly, 80 mg of amorphous PET (Goodfellow, Germany) was dissolved in 8 ml Hexafluoroisopropanol (1% v/v) at room temperature for 12 h. PET suspension was then added to the buret and the solution was added dropwise to ice cooled deionized (DI) water (100 ml) while constantly stirring. Stirring was continued for another 2 h. In order to remove bigger particles suspension was passed through filters (type 12, Cellulose membrane, 125 mm diameter, Roth, Germany). Subsequently, the organic solvent was removed from solution using a rotatory evaporator (Heidolph Instruments, USA) at elevated temperature and reduced pressure (50 °C, ~ 250 mbar). Next, the nanoparticles were allowed to settle in the cylinder for 2 h and the top 40 mL suspension was collected. Concentration of nanoparticles was determined gravimetrically by drying 2 mL of suspension in a pre-dried polymer pellets (3x) at 50 °C for 24 h and further weighing them to quantify the residual. The prepared solution was stored at room temperature, and it was stable for several weeks without settling.
6
Extraction of Azima tetracantha Leaves
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The Azima tetracantha Lam. was obtained from Athamangalam, Nagapattinam Distt, Tamilnadu, India, from August to September 2018. The herbarium was maintained in the Department of Botany, Annamalai University (Plant authentication number AUBOT 262). One hundred grams of crushed leaf materials were extracted in a soxhlet apparatus for 8 h with solvent MeOH (99.35%, Sigma Aldrich, Bangalore, India). Later the extracts were sieved, and the solvent was evaporated by a rotatory evaporator (Heidolph, Germany) under a concentrated temperature at 40 °C, and the extracts were kept at 4 °C for further examination.
7
Quantification of Free Fatty Acids in Milk
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The presence of free fatty acids (FFAs) in milk samples was determined following plasma application. Lipid extraction was carried out according to the method described by Lopez-Lopez, Castellote-Bargallo, and Lopez-Sabater (2001) with some modifications and standards used as previously described by (Lanciotti et al., 2006) . Dichloromethane-methanol (2:1) (Sigma, Munich, Germany) was added to milk and the mixture was mechanically agitated in a shaker (Hotech, Taipei, Taiwan) and then centrifuged at 3000 × g. After washing with sterile distilled water, the organic phase was filtered and the solvent was removed in a rotatory evaporator (Heidolph, Schwabach, Germany).
Lipids were extracted by methylation using n-hexane (Merck, Darmstadt, Germany) and 2 M potassium hydroxide in methanol (Merck, Darmstadt, Germany). After evaporation under N 2 flux, diazomethane (Sigma, Munich, Germany) was added. Fatty acid methyl ester analysis was carried and Wiley version 6 Mass spectral data base. The quantification of FFA level of milk samples was performed using C11:0 as an internal standard at concentration of 100 µL per 2.5 mg of fatty acid esters.
Lipids were extracted by methylation using n-hexane (Merck, Darmstadt, Germany) and 2 M potassium hydroxide in methanol (Merck, Darmstadt, Germany). After evaporation under N 2 flux, diazomethane (Sigma, Munich, Germany) was added. Fatty acid methyl ester analysis was carried and Wiley version 6 Mass spectral data base. The quantification of FFA level of milk samples was performed using C11:0 as an internal standard at concentration of 100 µL per 2.5 mg of fatty acid esters.
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