R 215 rotary evaporator

This research has been undertaken with the approval (Permit No. POS 248/2019/2020) of the South African Health Products Regulatory Authority to conduct, collect, possess, transport, and store cannabis plant, plant parts, and products for research purposes. The study was also conducted to collect cannabis plants in Lesotho under the permit (Permit #: 01/LS/2019/10/02-01).
The leaves of C. sativa were obtained from Mohale’s Hoek District, Lesotho (GPS coordinates: 30.333,776″S and 27.651,201″E). The plant was authenticated by the Geo Potts Herbarium at the University of the Free State, Bloemfontein 9300, South Africa, and assigned the voucher number BLFU MGM 0018. This was further verified in the plant list online database (http://www.theplantlist.org/tpl1.1/search?q=Cannabis+Sativa+L).
The leaves were air-dried and pulverized to dry powder before undergoing sequential extraction using solvents of increasing polarity, namely, hexane, dichloromethane (DCM), and ethanol, for 48 h with gentle agitation of 100 rpm at room temperature. Each solvent was decanted and concentrated in vacuo using an R-215 rotary evaporator (Buchi, Flawil, Switzerland). The extracts were collected in glass vials and stored in the dark at ambient room temperature.

The bacterial strain, stored at −80 °C in nutrient broth with 20% glycerol, was streaked onto nutrient agar (peptone 3 g·L⁻¹, beef extract 5 g·L⁻¹ and agar 15 g·L⁻¹ pH 7) plates and cultivated for 24 h at 28 °C. After 24 h of growth, bacterial cells were scraped off the plates into 10 L of sterile nutrient broth (peptone 3 g·L⁻¹ and beef extract 5 g·L⁻¹ pH 7) and incubated on a rotatory shaker at 28 °C and 180 rpm for 24 h.
Metabolic elicitors (released into the medium) were obtained by centrifuging the 10 L of N 21.4 culture at 2890× g during 20 min at 4 °C. Cells were discarded and the remaining supernatant was evaporated in a stove at 60 °C until obtaining 1 L. This concentrated supernatant was filtrated through a 0.2 µm nitrocellulose filter and extracted twice with a double volume of hexane (v/v). The extract was evaporated to dryness in a Buchi R-215 rotary evaporator at 50 °C [40 (link)]. The dry extract was weight (250 mg) and stored at 4 °C protected from light and humidity.
To obtain the control 1, the same procedure was followed as for extracting the metabolic elicitors from the bacterium, but while carrying out the entire process exclusively with the nutrient medium (peptone 3 g·L⁻¹ and beef extract 5 g·L⁻¹ pH 7), in the absence of bacterium.

Lebrunia neglecta organisms were collected at Puerto Morelos Reef Lagoon, by SCUBA diving at approximately 4 m depth. The sea anemones were kept in plastic bags with sea water, and carried to the laboratory. The extraction was conducted in a Ten Broeck homogenizer (Pyrex^®) using distilled water. Nematocyst discharge was monitored with a microscope in order to ensure the obtention of their active compounds. The crude extract was fractionated by size exclusion chromatography in a 118 × 1.5 cm Sephadex^® G-50 (Pharmacia Biotech) column using 0.3 M acetic acid as eluant at a 3 mL/min flux. Fractions of 15 mL were collected in conical tubes (Corning^®) and the absorbance was monitored at 280 nm using an Äkta prime plus chromatographer. The tubes corresponding to the same fraction were pooled and concentrated by reduced pressure in a Büchi R-215 rotary evaporator. Finally, the three fractions F1, F2 and F3 were lyophilized in a Labconco™ freeze dry system and stored at −70 °C until use.

Lycopene purified from red guava (LPG) was produced according to Amorim et al. [15 (link)]. Highly ripe red guava (Psidium guajava L.) from Parnaíba city, State of Piauí, Brazil, were subjected to dehydration with ethanol and extraction with dichloromethane under ultrasonic stirring. The extract was then filtered through quantitative filter paper and dried under reduced pressure (50 mbar) at room temperature in an R-215 rotary evaporator (Büchi Labortechnik, Flawil, Switzerland) under dim light. After that, purified lycopene was obtained by crystallization at −20 °C and washing with ethanol and chloroform. The isolated lycopene was stored at −80 °C.

Turbina oblongata was collected from Eastern Cape Province, South Africa (GPS coordinates: 32°44′20.1″S 26°54′58.3″E). The plant sample was identified and authenticated at the Geo Potts herbarium (BLFU), University of the Free State (specimen voucher number: BLFU MGM0019).
The plant sample was air-dried after washing and was blended to fine powder. The powdered sample (100 g in a 1:5 mass:solvent ratio in ml) was subjected to sequential extraction using solvents of increasing polarity vis-à-vis hexane, dichloromethane (DCM), methanol and dichloromethane (MeOH:DCM; 1:1, v/v) and methanol (MeOH). Each extraction was carried out at ambient room temperature for 24 h. After extraction, each extract was filtered and subsequently concentrated in vacuo with an R–215 rotary evaporator (Buchi, Switzerland). Concentrated samples were collected into glass vials and stored at 4 C.

Neutrophils from patients with HTN and NTI (1 × 10⁷) were lysed in 100 μL of 2× lysis buffer (0.5% NP-40, 2 mM Na₃VO₄, PSMF) in the presence of protease inhibitor cocktail, diluted to 1000 μL with PBS, and processed for lipid extraction, as previously described [42 (link)]. In brief, neutrophil suspensions were extracted in 3 mL of chloroform:methanol (2:1, v:v) by vortexing for 2 min, centrifuged for 5 min at 2000× g, and the chloroform fraction was evaporated to dryness under reduced pressure at room temperature using a BÜCHI R-215 Rotary Evaporator coupled to a BÜCHI V-700 Vacuum Pump and cold-water recirculating BÜCHI Chiller F-105 (BÜCHI Labortechnik AG, Flawil, Switzerland). The dried extracts were diluted in 1 mL of methanol for analysis.

Cannabis sativa leaves were obtained from Mohale’s Hoek District, Lesotho (GPS coordinates: −30.333776″S and 27.651201″E). They were authenticated by the Geo Potts Herbarium at the University of the Free State, Bloemfontein 9300, South Africa and assigned the voucher number, BLFU MGM 0018. The leaves were pulverized to dry powder, after air drying to a constant weight.
The powdered samples were thereafter sequentially extracted with solvents of increasing polarity vis-à-vis hexane and dichloromethane (DCM) for 48 h with mild agitation of 100 rpm at room temperature. The solvents were respectively decanted and concentrated in vacuo using an R–215 rotary evaporator (Buchi, Switzerland). The extracts were collected in glass vials and stored in the dark at ambient room temperature for further ex vivo studies.