Chloroform

Chromatographic separation was performed by preparative TLC Silica gel 60 F254 (0.5 mm thick) (Loba Chemie Pvt. Ltd., Mumbai, India). n-Hexane, chloroform, ethyl acetate and methanol (Loba Chemie Pvt. Ltd., Mumbai, India), trisodium citrate (BDH Chemicals Ltd., London, UK), and Giemsa (ESJAY Chemicals, Maharashtra, India) were all used as received. Pure chloroquine phosphate supplied by Ethiopian Pharmaceuticals Manufacturing Sh. Co. (EPHARM, Addis Ababa, Ethiopia) was used as a reference drug.

Hydrochloric acid 35.4% (Loba Chemie, India), sodium hydroxide (Loba Chemie Pvt. Ltd.), ammonium acetate 96% (Loba Chemie, India), methanol 99.8% HPLC grade (Loba Chemie, India), ethanol 96% (Fine Chemicals, Ethiopia), sodium chloride 99.5% (Loba Chemie, India), chloroform (Loba Chemie, India), acetone (Loba Chemie, India), and sulphuric acid (Loba Chemie, India) were purchased from local markets. All chemicals used in this experiment were analytical grade chemicals.

AZA was procured as a gift sample from Micro Labs Pvt. Ltd. (Bangalore, Karnataka, India). Lecithin, cholesterol and 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) (MTT) was purchased from HiMedia laboratories Pvt. Ltd. (Mumbai, India). Chloroform was purchased from Lobachemie Pvt Ltd. (Mumbai (India) and deionized water was purchased from Milli-Q system (Millipore, Bedford, Mumbai, India). Fluorescein isothiocynate (FITC) and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma Aldrich (Mumbai, India). All other solvents and chemicals were of analytical grade and obtained from other local supplier.

Biocon Pvt Ltd., Bangalore, India generously provided SIM. Pluronic F-127, chloroform, dextrose and xanthan gum were procured from Loba Chemie Pvt Ltd., Mumbai, India. All other chemicals used were of analytical grade.

Chara vulgaris was collected from southern part of Khulna, Bangladesh, chosen on the basis of higher accessibility. It is found throughout the year but higher amount found from May to September. Total lipid content of Chara vulgaris is 73.2 mg/g of dry weight (Dembitsky and Rezanka 1993 ). Total lipid extraction from algae performed by chemical extraction methods, relying on the Chloroform–Methanol solvent system, based on the Bligh and Dyer method (Bligh and Dyer 1959 (link)). Sodium Chloride solution was added to facilitate oil extraction from algae (Axelsson and Gentili 2014 ). A slight modification of this process was done to facilitate the lipid extraction from Chara vulgaris. Lipid found from this step was then used for the transesterification reaction to produce biodiesel. Chloroform (Loba Chemie, Mumbai, India), Sodium Chloride (Merck, Mumbai, India), Methanol (Merck KGaA, Darmstadt, Germany) and Sodium hydroxide (Merck, Mumbai, India) used for the oil extraction and transesterification process.