Tween 80

EO-ACs activity against planktonic cells of both the Cryptococcus species was performed by standard broth microdilution method recommended by Clinical and Laboratory Standards Institute (CLSI, 2008 ) reference protocols M27-A3, with a modification of replacing RPMI 1640 medium by Yeast Nitrogen Base (YNB, HiMedia, India). Planktonic cells grown in SDB were harvested at exponential phase, washed with sterile 1X phosphate-buffered saline (PBS pH 7, 0.1M) and re-suspended in YNB medium at a density of 1–5 × 10⁴ cells/mL. Serially double diluted concentration of EO-ACs/drugs (0–1,024 μg/mL) were added in 96-well microtiter plates to provide 0.5–2.5 × 10⁴ cells/mL in 200 μL working volume. YNB medium with 1% DMSO plus 10% mineral oil (HiMedia) was added to control wells (Fontenelle et al., 2007 (link)). Tween-80 (HiMedia, India) at 0.05% (v/v) final concentration was added in all assays to enhance EO-ACs solubility. The plates were then incubated at 30°C for 24 h without shaking. After incubation, the growth of cells was measured by microtiter plate reader (SpectraMax, Molecular Devices, USA) at 530 nm and Minimum inhibitory concentration (MIC₈₀) which reduces 80% cell growth as compared to control (without EO-ACs/drugs) was determined.

MTB (clinical strain) was used for the study. Culture was maintained on Lowenstein-Jenson (LJ) slant before experiment strain was freshly streaked on agar plate after 21 days of full growth. It was again inoculated in Middlebrook 7H9 broth for 14 days as a primary (1^o) culture. Starter culture H₃₇Rv untreated or treated with INH drug at subinhibitory conc. of 0.5 μg/ml was inoculated with primary (1^o) culture of 0.1 OD₆₀₀ in a screw cap flask containing 10 mL of Middlebrook 7H9 broth supplemented with 10% albumin/dextrose/catalase (BD Difco), 0.2% glycerol (Fischer Scientific), and 0.05% Tween-80 (Himedia) in 100 mL flasks (Schott Duran). The flasks were placed at 37°C for 14 days or till its exponential phase with agitation.

XH (Xantho Flav) was gifted by Simon H. Steiner, Hopfen, GmbH (Mainburg, Germany). Compritol E ATO (CE), Precirol ATO5 was gifted by Gattefosse, (Mumbai, India). Lipoid LIPOID E 80SN (LE-80), Lipoid S 75, and Phospholipon 90 H were gifted by Lipoid GmbH (Germany). Pluronic F-68 and tween 80 were purchased from Hi-Media laboratories (Mumbai, India). Monemul-20 was gifted from Mohini chemicals, Mumbai, India. Glyceryl monostearate (GMS) and carnauba wax were purchased from Sigma-Aldrich (New Delhi, India). Palmitic acid and cetyl alcohol were purchased from Lobachemie Pvt Ltd. (Mumbai, India). Stearic acid was purchased from Qualigen fine chemicals (Mumbai, India). Sephadex G-25, dialysis bag (14–15 kDa) was purchased from GE Healthcare, Hyderabad. Methanol and orthophosphoric acid (OPA) of HPLC grade were purchased from Molychem (Mumbai, India). Water was passed through Milli Q filter to prepare the mobile phase.

Zinc nitrate hexahydrate (Zn (NO₃)₂.6H₂O), DON (purity 98% TLC and CAS number: 51481-10-8), Dulbecco’s phosphate buffer saline pH 7.4 (DPBS), lipid peroxidation assay kit, and ZEA (purity 99% HPLC and CAS number: 17924-92-4) were purchased from Sigma-Aldrich, India. Peptone, Tween 80, CZA, CZB, PI, sterile gauze, DCFH-DA, syringe filters (0.45 μm), double-layered muslin cloth, and Whatman no.1 filter papers were obtained from HiMedia, India. Immuno-affinity columns specific for DON and ZEA were obtained from Vicam, Waters, United States. All other chemicals and reagents were of AR grade and purchased from Merck Millipore, India. The plastic ware was purchased from Nunc, India. Glassware used were washed with 2% sodium hypochlorite solution and maintained in sterile condition.

Culture media reagents (Müeller Hinton Broth—MHB; Müeller Hinton agar—MHA), ceftazidime (CAZ), curcumin from Curcuma longa L. (CUR), and surfactants (Tween 80 and Span 80) were obtained from Himedia® and Sigma-Aldrich®. All solvents (Dimetilsulfoxide—DMSO, saline solutions) were purchased from Merck®^.The products are as the following:
ceftazidime: Formula—C22H22N6O7S2, Molecular weight—636.65 g/mol, CAS number—78,439–06-2, concentration—100%). curcumin: Formula—C21H20O6, Molecular weight—368.38 g/mol, CAS number—458–37-7, Purity (HPLC)— ≥ 65%.

Serum samples of TB patients were collected from Government Medical College and Hospital (GMCH), Sec-32, Chandigarh, India. The collection of human serum samples was approved by the Institute Ethics Committee of GMCH, Chandigarh, India and written consent was obtained from all participants. All experiments were conducted in accordance with the Ethical Guidelines for Biomedical Research on Human Subjects, Indian Council of Medical Research and Government of India.
E. coli DH5α and Mycobacterium smegmatis mc²155 were procured from IMTECH-MTCC, Chandigarh. The E. coli- Mycobacterium shuttle vector (pVV16) was a kind gift from Dr. Stephen Cannan, France. The middle brook 7H9 broth, glycerol, and tween-80 were purchased from Hi Media, India. The supplement OADC was purchased from BD bioscience (India). RPMI media and Fetal bovine serum (FBS) were purchased from Sigma chemicals (USA).