Source Name: 2018_Ramalho
Literature Information
| Literature Title | Synthesis, Racemic X-ray Crystallographic, and Permeability Studies of Bioactive Orbitides from Jatropha Species |
| Doi | 10.1021/acs.jnatprod.8b00447 |
| Research Group |
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| Data Number | 10 |
| Minimum Molecular Weight | 767.0 |
| Maximum Molecular Weight | 851.0 |
Assay Information 1
| Assay Type | PAMPA |
| Permeability Type | logPapp |
| Membrane Measurement Direction (Exclude PAMPA) | - |
| Assay Detail | A 96-well filter plate coated with lipids (BD Gentest precoated PAMPA plates) was used. A 300 μL solution of 50 μM peptide (prepared by diluting the 5 mM DMSO stock solutions with Hank’s balanced saline solution [HBSS]) was added to each well of the donor (bottom) plate, and 200 μL of HBSS was added to each acceptor well (top plate). The acceptor plate was then placed on top of the donor plate so that the artificial membrane was in contact with the buffer solution below. A lid was placed on the acceptor plate, and the system was covered and left for 5 h at room temperature. Donor well solutions before (t = 0h) and after (t =5h) incubation as well as acceptor well solutions were diluted in MeCN (1:1) and analyzed by LC-MS through a 5 μm C18 column using the selected ion monitoring (SIM) mode. Each PAMPA measurement was performed in triplicate. Atenolol and quinidine were used as controls of known and varied permeability. |
Assay Information 2
| Assay Type | Caco2 |
| Permeability Type | logPapp |
| Membrane Measurement Direction (Exclude PAMPA) | Apical to Basolateral (AB) |
| Assay Detail | The permeability was evaluated using an accelerated 6-day Caco-2 permeability model as described previously. Briefly, cells were seeded onto each membrane of the Transwell permeable support 12-well plate (Corning) insert to give 2.5 x 10^5 cells/well. The individual feeding tray wells received 0.75 mL of assay medium (with 1% penicillin/streptomycin replaced with 0.4 μg/mL puromycin) in the top compartment and 1.5 mL in the bottom compartment. The assay medium was changed every second day. After a 6-day cell culture of Caco-2 cells, the medium in the top compartment was replaced with peptide solutions at 50 μM and incubated for 90 min at 37 °C, after which concentrations in the apical and basolateral compartments were quantified using LC-MS (Qstar Elite, AB Sciex Australia) to calculate compound flux in the apical-to-basolateral direction. Lucifer yellow and trans-epithelial electrical resistance (TEER) measurements were used to validate membrane integrity of the Caco-2 cell monolayer. Atenolol and quinidine were used as controls of known and varied permeability. |