Dimethyl sulfoxide (dmso)

All chemicals and reagents were of analytical grade and used without any treatment. 1,10-phenanthroline (BDH chemical Ltd., Poole, England), chrysin (Sigma Aldrich), chromium chloride hexahydrate (CrCl₆.6H₂O), dimethyl sulfoxide (DMSO), and dimethylformamide (DMF) were purchased from Loba Chemie Pvt. Ltd. (Mumbai, India). Triethylamine, NaHCO₃, Mueller–Hinton agar, methanol (MeOH), and diethyl ether were purchased from (Alpha Chemika, India). Ascorbic acid and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich.

Lipopolysaccharide (LPS, from E coli, serotype 0111:B4), phenylephrine hydrochloride, 5 -N-ethylcarboxamidoadenosine (NECA, adenosine analogue), acetylcholine (ACh), N_ω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) (Sigma-Aldrich Co., St. Louis, MO, United States), losartan potassium, pioglitazone hydrochloride (PHARCO Pharmaceutical Co., Alexandria, Egypt), thiopental sodium (Biochemie, Vienna, Austria) and heparin (5000 IU/mL; Nile Pharmaceutical Co., Egypt) were purchased from commercial vendors. ACh and NECA were freshly prepared in distilled water and dimethyl sulfoxide (Loba Chemie Pvt Ltd, India), respectively. LPS, heparin and thiopental were dissolved in saline.

First, 20, 50, and 100 mM stock solutions of TQ (Acros Organics, Geel, Belgium code:305070010, GC: 98.6%) were dissolved in dimethyl sulfoxide (DMSO) (Loba Chemie, Mumbai, India) and stored at −80 °C. The TQ stock solutions were diluted in fresh cell culture media into (20, 50, and 100 µM) just before use. Note that the percentage of DMSO was 0.1% in order to not be toxic to cells.

The antimicrobial activities of the extracts were first screened for their inhibitory zones using the agar disk diffusion method [31 ]. A sterile cotton swab was dipped into the suspension. Then, the inoculum was spread evenly over the entire MHA agar surface. A total of 20 µL of the extracts, which worked as antimicrobial agents, was dropped on filter paper discs with a diameter of 6 mm, and the filter paper discs were placed on the MHA plates. Discs impregnated with normal saline solution and DMSO (Loba Chemie Pvt. Ltd., Mumbai, India) were used as a negative control. Tetracycline (Oxoid, Basingstoke, UK) and chloramphenical (Oxoid, UK) were applied as control antibiotics for comparison. The plates were incubated at 35 ± 1 °C for 18 ± 2 h in an incubator (Benchmark, H2200-H, Suzhou, China). The inhibition zone’s diameter was measured using Vernier calipers (Total Tools, Suzhou, China), including the 6 mm diameter filter paper discs (Whatman, Hangzhou, China). The effectiveness of the extracts on antimicrobial activity increased with a larger inhibition zone of bacteria. Each antibacterial test was conducted with three replicates. The extracts with the highest solubility from different solvents in DMSO were selected for the determination of the inhibition zone.

The chemicals that were used in this study includes: dimethyl sulphoxide (DMSO) (LobaChemie.Pvt.Ltd, India), 5% hematocrit (human type A-positive erythrocytes, Italy), RPMI (Roswell Park Memorial Institute) 1640 medium (EuroClone, Celbio), 1% AlbuMax (Invitrogen, Milan, Italy), 0.01% hypoxanthine, 20 mM HEPES, 2 mM glutamine, absolute methanol (ALPHA CHEMIKA, India), the standard drug Chloroquine. In addition, the following instruments and materials: grinder (Hi-speed Multifunctional Grinder, Shanghai Yuan WO Industrial and Trade Co.Ltd.Yongkang City), maceration jar, orbital shaker (Stuart, UK), vacuum pump (PoongilCommercial.Co. Ltd, Yongsan-Gu.Seoul, Korea), muslin cloth, Whatman filters paper No.1, collecting flask, drying oven (GENLAB WIDNES, England), refrigerator,light microscope (OPTIKA, ITALY), frosted microscopic slides, measuring cylinder, 96-well flat-bottomed microplates, incubator,and spectrophotometer were used to perform the study.