Biochemical Methods of Studying Hepatotoxicity
Robert G. Meeks, Steadman D. Harrison, Richard J. Bull in Hepatotoxicology, 2020
Add 0.5 ml tris buffer, 0.2 ml MgCl2, 0.025 ml NADPH, and 0.025 ml benzo(a)pyrene to a test tube. Incubate the reaction mixture at 37°C. Start the reaction with 0.25 ml microsomal sample containing 0.5 to 0.8 mg protein. Incubate at 37°C for 30 min. Stop the reaction by adding 1 ml of cold acetone. Maintain proper blanks by adding acetone prior to incubation. After the addition of acetone, add 3.25 ml hexane and incubate the mixture with shaking at 37°C for 10 min. Take 1 ml of organic phase and extract with 3.0 ml of 1 N NaOH. Determine the concentration of extracted, hydroxylate benzo(a)pyrene in the alkali phase spectrofluorometrically with excitation at 396 nm and emission at 522 nm. Prepare standard graph by using quinine sulfate or 3-hydroxybenzo(a)pyrene. Prepare standard solutions in 0.1 N H2SO4. Calculate the sample values from the standard graph. One unit of enzyme activity can be defined as that amount catalyzing the formation of hydroxylated product causing fluorescence equivalent to that of 1 pmole of 3-hydroxybenzo(a)pyrene in a 30 min incubation at 37°C.
Synthetic Approaches to Inhibitors of Isoprenoid Biosynthesis
Peter Grunwald in Pharmaceutical Biocatalysis, 2019
A series of N-bisphosphonates bearing either the nitrogen-containing furoxan (1,2,5-oxadiazole 2-oxide) or the related furazan (1,2,5-oxadiazole) systems in the lateral chain were prepared in discrete yields by using trimethylphosphite as P-reagent (Scheme 2.17) (Lolli et al., 2010). Reagents and conditions: (i) 1,5-pentanediol, NaOH 50%, THF. (ii) Jones reagent, acetone, 0°C to rt. (iii) SOCl2. (iv) P(OMe)3, dry THF, 0°C to rt. (v) HPO(OMe)2, Et2NH, dry THF, 0°C to rt; (vi) TMSBr, CH2Cl2; then MeOH, 0°C to rt.
Role of Process Standardisation in Development of Natural Products
Dilip Ghosh, Pulok K. Mukherjee in Natural Medicines, 2019
Anthocyanins in plants occur as glycosides of six common anthocyanidins including cyanidin, delphinidin, petunidin, peonidin, pelargonidin and malvidin, which give a different colour spectrum of anthocyanins ranging from red to purple (Figure 6.1). The structure of anthocyanins is polar, so they can be extracted using various organic polar solvents. The common solvents used for anthocyanin extraction are methanol, ethanol, acetone, water or a mixture of these solvents. Methanol is efficient in terms of anthocyanin extraction, but its toxicity limits its use, especially when the anthocyanins are meant to be used in foods. Acetone is less toxic than many other solvents and allows an efficient and more reproducible extraction. Using acetone to extract plant anthocyanins can avoid the interference from pectin that is generally dissolved in alcohol or water. Acetone requires lower temperature for evaporation after extraction. Acid is added into the extraction solvent to keep anthocyanin in its stable flavylium cation form at an acidic pH that is generally lower than pH 2. Still, highly acidic solution may cause partial hydrolysis of acyl moieties in acylated anthocyanins. Hydrochloric acid was widely used in the past, but more recently weaker acids such as formic acid, tartaric acid or citric acid have become more commonly used. After solvent extraction, solid phase purification is used to purify anthocyanin from other compounds such as organic acids that are co-present in the crude extract.
PLGA sustained-release microspheres loaded with an insoluble small-molecule drug: microfluidic-based preparation, optimization, characterization, and evaluation in vitro and in vivo
Published in Drug Delivery, 2022
Yue Su, Jia Liu, Songwen Tan, Wenfang Liu, Rongrong Wang, Chuanpin Chen
The effects of dichloromethane to acetone (D/A) volume ratio on EE% and DL% were investigated when BCL concentration was 7.5 mg/mL and PLGA concentration was 1 wt%. As shown in Figure 3(A), with the decrease of the D/A volume ratio, the EE% and DL% of microspheres first increased and then decreased. When the D/A volume ratio was 3:1, DL% and EE% of microspheres were the highest. BCL was slightly dissolved in DCM, and DL% and EE% of the microspheres prepared were low when only DCM was used as the solvent. When acetone was added, DL% and EE% increased as the ratio of the D/A volume ratio decreased, because acetone was beneficial to the dissolution of BCL. However, acetone is a miscible solvent with water. When the volume of acetone increased to a certain level, part of the drug could diffuse into the water phase with acetone, resulting in the reduction of the drug content in the microspheres.
Occupational exposure assessment with solid substances: choosing a vehicle for in vitro percutaneous absorption experiments
Published in Critical Reviews in Toxicology, 2022
Catherine Champmartin, Lisa Chedik, Fabrice Marquet, Frédéric Cosnier
Except when seeking to mimic exposure to aqueous solutions of toxicants, water should be used with caution as it is a well-known enhancer, interacting with both lipids and corneocytes in the SC. Considering the various effects of ethanol, it is not recommended for use neat, and it should particularly be avoided when studying skin metabolism. There is little advantage to using it in combination with water. The use of a volatile solvent may be an alternative means to directly deposit solid substances. In this case, a small deposit volume should be used to allow rapid evaporation and limit the effect on the skin barrier. Acetone is preferable to ethanol. Although more realistic, the use of artificial sebum requires first to decide on its composition, and then to develop a technique to evenly apply a small volume to the skin, to accurately mimic its secretion in vivo. After all these preliminary steps, it will be necessary to assess the effects of the artificial sebum on the skin. For artificial sweat, it is currently difficult to conclude, given the paucity of data and the lack of consensus composition. However, we recommend that the solution be isotonic, including artificial sweat or NaCl 0.9% in water, rather than ultrapure water in its composition.
Preparation and evaluation of polyphenol derivatives as potent antifouling agents: addition of a side chain affects the biological activity of polyphenols
Published in Biofouling, 2022
Xuan Wang, Xiaohui Jiang, Liangmin Yu
Compared with ethanol and DMSO, acetone has no antibacterial activity at the tested concentration, so it is the most suitable solvent when detecting biological performance. The antibacterial activities of the polyphenol derivatives are shown in Figures 2 and 3. After cultivation for 18 h, the bacterial colonies with polyphenol derivatives were significantly less abundant than those of the control (Figure 2), and their antibacterial rates were > 88% at 20 μg ml−1 (Figure 3), indicating that polyphenol derivatives strongly inhibited the growth of the bacteria. In addition, their antibacterial activity was substantially higher than that of polyphenols. The polyphenol derivatives containing phenolic hydroxyl and amide groups showed similar antibacterial activities due to their similar structures; among these compounds, a3 and b3, containing chlorine atoms, showed the highest antibacterial activity, indicating that the addition of chlorine atoms could improve the antibacterial activity.
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