Moringa oleifera leaves were harvested from the Botanical Garden, University of Ibadan, Nigeria. They were identified and authenticated at the herbarium of the Department of Botany, University of Ibadan, where a voucher specimen was also deposited (Voucher number: UIH-22770). The leaves were air-dried and milled into coarse powder using an electric blender. In an initial de-fatting process, about 1.6 kg of the grounded sample was weighed and transferred into a glass container with 6L of n-hexane. This was stirred regularly every two hours over a 72 h period. About 1.5 kg of the defatted sample was transferred into a glass container and 6L of a methanol-water (80:20 v/v) was added and allowed to stay for another 72hours, while stirring at intervals. The solvent (now containing the extract) was filtered using Whatman No. 1 filter paper. The process was repeated with another 4L of methanol-water solvent mixture. The combined filtrate was then concentrated with the aid of rotary evaporator (Heidolph Laborota 400; Model 517-01002-002 Germany) at 40°C, after which the resulting extract was further concentrated using a vacuum oven at 40°C with a pressure of 700 mmHg.
Laborota 400
Manufactured by Heidolph
Sourced in Germany
The Heidolph Laborota 400 is a rotary evaporator designed for laboratory use. It is capable of evaporating solvents from samples, concentrating solutions, and performing other distillation processes. The Laborota 400 features a digital control panel, adjustable rotation speed, and a water bath for temperature control.
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Lab products found in correlation
Diaphragm vacuum pump dc 4, by Heidolph (3 mentions)
Avanti mini extruder, by Avanti Polar Lipids (1 mentions)
D sucrose, by Merck Group (1 mentions)
Resomer rg 752h, by Merck Group (1 mentions)
Sodium hyaluronate ha, by Lifecore Biomedical (1 mentions)
Milli q water, by Merck Group (1 mentions)
D glucose, by Merck Group (1 mentions)
D mannitol, by Merck Group (1 mentions)
Sodium taurodeoxycholate, by Merck Group (1 mentions)
Sepharose cl 4b, by Merck Group (1 mentions)
Benzyl alcohol, by Merck Group (1 mentions)
Pluronic f68, by Merck Group (1 mentions)
W717 plus autosampler, by Waters Corporation (1 mentions)
2996 pda detector, by Waters Corporation (1 mentions)
Cremophor rh60, by BASF (1 mentions)
Ethyl acetate, by Merck Group (1 mentions)
Cholesterol, by Merck Group (1 mentions)
Miglyol 812n, by Sasol (1 mentions)
Phalloidin atto 488, by Merck Group (1 mentions)
Sodium 1 heptanesulfonate, by Merck Group (1 mentions)
7 protocols using laborota 400
1
Moringa oleifera Leaf Extraction and Characterization
2
Liposome Preparation and Characterization
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Ph (100% saturated phosphatidylcholine) was dissolved in CHCl3:MeOH (2:1 v/v) to prepare a 10 mg/mL stock solution. Liposome suspensions were prepared in molar ratios of 10:0, 7:3, 5:5, and 0:10 TEL:Ph. These suspensions were transferred to 10 mL round bottom flasks and evaporated at 45 °C 300 mbar with a rotary evaporator (Heidolph, Laborota 400, Schwabach, Germany) to obtain a lipid film. First, the thin film was hydrated with bidistilled water containing 0.9% NaCl. The solution was sonicated with a probe-type sonicator with an increased energy input (G. Heinemann Ultraschall und Labortechnik Schwöbosch, Germany), and a homogeneous suspension of the liposomes in water was obtained. Sonication was applied for 8 min (30 s sonication followed by 30 s rest). After sonication, liposomes were filtered with syringe filters through a pore size of 0.2 µm. Finally, all the liposome suspensions were extruded 21 times through a 0.1 µm polycarbonate membrane filter by a mini extruder device (Avanti, Hamburg, Germany).
3
Preparation of Small Unilamellar Liposomes
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Liposomes were prepared using the film hydration method [18 (link)]. Stock solutions of lipids were prepared by dissolving the lipids in a CHCl3 : MeOH (2 : 1 v/v) solution. Required amounts of lipid solutions were mixed in a 5 mL RBF and evaporated to dryness using a Laborota 400 rotary evaporator from Heidolph Instruments (Schwabach, Germany) creating a thin lipid layer on the wall of the flask (under vacuum, 280 rpm/min). The films were hydrated with 20 mM HBS buffer to obtain a lipid concentration of 6 mg/mL, and the flasks were placed in a bath sonicator and allowed to equilibrate for 5 minutes. Then, the flasks were sonicated for 2 minutes until the lipid film was fully reconstituted. Liposomal suspension was further extruded through 200 nm and 100 nm polycarbonate membranes (Whatman) using an Avanti Mini Extruder (Avanti Polar Lipids) to obtain small unilamellar liposomes. For cell-culture experiments, liposomes were filter-sterilised through a 0.22 μm syringe filter and stored at 4°C up to two weeks.
4
Synthesis and Characterization of Hyaluronic Acid-Phospholipid Conjugates
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All the phospholipids, cholesterol (CHOL), PLGA 50:50 (Resomer® RG 502 H, 7–17 kDa), PLGA 75:25 (Resomer® RG 752 H, 4–15 kDa), PEG2000-PLGA 50:50 (PLGA 11.5 kDa), D-(+)-glucose, D-(+)-sucrose, D-(+)-trehalose, D-(+)-mannitol (purity minimum 95%) and solvents (analytical grade) were purchased from Merck (Milan, Italy). Sodium hyaluronate (HA) (4.8 kDa or 14.8 kDa) was purchased from Lifecore Biomedical (Chaska, MN, USA). The compound 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) was conjugated to 4.8 kDa or 14.8 kDa HA (HA4.8-DPPE or HA14.8-DPPE) using the method described by Arpicco et al. [30 (link)]. Filtered MilliQ® water (Millipore, Merck) was used. Solvents were evaporated using a rotating evaporator (Heidolph Laborota 400, Heidolph Instruments, Schwabach, Germany) equipped with a vacuum pump (Diaphragm Vacuum Pump DC-4).
5
Lipid Nanoparticle Formulation and Characterization
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All the phospholipids were provided by Avanti Polar Lipids and distributed by Sigma-Aldrich (Milan, Italy). Miglyol 812N was a gift from Sasol (Witten, Germany). Poly(lactide-co-glycolide) (PLGA) 75:25 (Resomer RG 752 H), trilaurin, ethyl acetate, benzyl alcohol, sodium taurodeoxycholate, Pluronic F68, Sepharose CL-4B, cholesterol and all other reagents were obtained from Sigma-Aldrich. Cremophor RH 60 (PEG-60 hydrogenated castor oil) was purchased from BASF (Ludwigshafen, Germany). All the solvents used were of analytical grade and purchased from Carlo Erba Reagenti (Milan, Italy). Solvent evaporation was carried out using a rotating evaporator (Heidolph Laborota 400, Heidolph Instruments, Schwabach, Germany) equipped with a vacuum pump (Diaphragm Vacuum Pump DC-4). TLC analyses were performed on silica gel aluminium plates (Macherey-Nagel, thick 25 µm, F254). HPLC analyses were carried out on a Waters instrument made up of 1525EF binary pump, W717 plus auto-sampler and 2996 PDA detector (Waters, Milan, Italy).
6
Biodegradable PLGA Nanoparticle Formulation
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PLGA 75:25 (Resomer® RG 752 H, Mw = 4–15 kDa) (analytical grade), SC, PVA (Mw = 31–50 kDa, 98–99% hydrolyzed), PTM isethionate (PTM-I), dimethyl sulfoxide (DMSO), phosphoric acid, sodium hydroxide and sodium 1-heptanesulfonate were purchased from Merck (Milan, Italy). All the solvents used were of analytical grade or HPLC grade and were purchased from Carlo Erba Reagenti (Milan, Italy). Ultrapure water used for the buffers was obtained from a Milli-Q® Plus Purification System (Merck Millipore, Vimodrone Milan, Italy). Solvent evaporation was carried out using a rotating evaporator (Heidolph Laborota 400, Heidolph Instruments, Schwabach, Germany) equipped with a vacuum pump (Diaphragm Vacuum Pump DC-4). Lyophilization was performed with a LyoQuest-85® freeze drier (Azbil Telstar Technologies, Barcelona, Spain). C2C12 myoblasts, an immortalized murine cell line, were purchased from ECACC 91031101. Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), glutamine, amphotericin B and penicillin-streptomycin were purchased from Gibco, Thermo Fisher Scientific (Waltham, MA, USA). Thiazolyl Blue Tetrazolium Bromide (MTT solution), Phalloidin-Atto 488, Phalloidin-Atto 594 and Alexafluor 488 were obtained from Merck.
7
Methanol Extraction of Plant Bioactive Compounds
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Two kilograms of the dried seed sample was transferred into a glass container; 7.5 L of pure methanol was added, then stirred every 2 h with a glass rod and allowed to stand for 72 h. The solvent (now containing the extract) was collected using a muslin bag. The filtrate was further filtered using Wattman No. 1 filter paper. This process was repeated twice with another 5.0 L of pure methanol added each time to the chaff. The combined filtrate was then concentrated with the aid of a rotary evaporator (Heidolph Laborota 400; Heidolph Instruments, Kelheim, Germany) set at 40°C, after which the sample was further concentrated using a vacuum oven set at 40°C. The dried extract was weighed and the percentage yield was calculated.
Both qualitative and quantitative phytochemical screening of the plant extract were performed using standard procedures.
Both qualitative and quantitative phytochemical screening of the plant extract were performed using standard procedures.
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