Ferrous chloride

Determination of total phenolic content (TPC): Amount of TP were assessed using the Folin-Ciocalteu reagent [25 ]. Briefly, the crude extract (50 mg) was mixed with Folin-Ciocalteu reagent (0.5 mL) and deionized water (7.5 mL). The mixture was kept at room temperature for 10 min, and then 20% sodium carbonate (w/v, 1.5 mL) was added. The mixture was heated in a water bath at 40 ^oC for 20 min and then cooled in an ice bath; absorbance was read at 755 nm using a spectrophotometer (U-2001, Hitachi Instruments Inc., Tokyo, Japan). Amounts of TP were calculated using gallic acid calibration curve within range of 10-100 mgL^-1(R² = 0.9986). The results were expressed as gallic acid equivalents (GAE) g/100g of dry plant matter. All samples were analyzed thrice and the results averaged. The results are reported on dry weight basis (DW).
Determination of total flavonoid contents (TFC): The TFC were measured following a previously reported spectrophotometric method [26 (link)]. Briefly, extracts of each plant material (1 mL containing 0.1 mg/mL) were diluted with water (4 mL) in a 10 mL volumetric flask. Initially, 5% NaNO₂ solution (0.3 mL) was added to each volumetric flask; at 5 min, 10% AlCl₃ (0.3 mL) was added; and at 6 min, 1.0 M NaOH (2 mL) was added. Water (2.4 mL) was then added to the reaction flask and mixed well. Absorbance of the reaction mixture was read at 510 nm. TFC were determined as catechin equivalents (g/100g of dry weight). Three readings were taken for each sample and the results averaged.
Determination of reducing power: The reducing power of the extracts was determined according to the procedure described earlier [27 ], with a slight modification. Concentrated extract (2.5-10.0 mg) was mixed with sodium phosphate buffer (5.0 mL, 0.2 M, pH 6.6) and potassium ferricyanide (5.0 mL, 1.0%); the mixture was incubated at 50 ^oC for 20 min. Then 10% trichloroacetic acid (5 mL) was added and the mixture centrifuged at 980 g for 10 min at 5 °C in a refrigerated centrifuge (CHM-17; Kokusan Denki, Tokyo, Japan). The upper layer of the solution (5.0 mL) was decanted and diluted with 5.0 mL of distilled water and ferric chloride (1.0 mL, 0.1%), and absorbance read at 700 nm using a spectrophotometer (U-2001, Hitachi Instruments Inc., Tokyo, Japan). All samples were analyzed thrice and the results averaged.
DPPH^. scavenging assay: 1, 1^’–diphenyl–2-picrylhydrazyl (DPPH) free radical scavenging activity of the extracts was assessed using the procedure reported earlier [28 (link)]. Briefly, to extract (1.0 mL) containing 25 μg/mL of dry matter in methanol, freshly prepared solution of DPPH (0.025 g/L, 5.0 mL) was added. Absorbance at 0, 0.5, 1, 2, 5 and 10 min was measured at 515 nm using a spectrophotometer. The scavenging amounts of DPPH radical (DPPH^.) was calculated from a calibration curve. Absorbance read at the 5th min was used for comparison of radical scavenging activity of the extracts.
Determination of antioxidant activity in linoleic acid system: The antioxidant activity of the tested plant extracts was also determined by measuring the oxidation of linoleic acid [28 (link)]. Five mg of each plant extract were added separately to a solution of linoleic acid (0.13 mL), 99.8% ethanol (10 mL) and 0.2 M sodium phosphate buffer (pH 7, 10 mL). The mixture was made up to 25 mL with distilled water and incubated at 40 ^oC up to 360 h. Extent of oxidation was measured by peroxide value applying thiocyanate method as described by Yen et al. [27 ]. Briefly, ethanol (75% v/v, 10 mL ), aqueous solution of ammonium thiocyanate (30% w/v, 0.2 mL), sample solution (0.2 mL) and ferrous chloride (FeCl₂) solution (20 mM in 3.5% HCl; v/v, 0.2 mL) were added sequentially. After 3 min of stirring, the absorption was measured at 500 nm using a spectrophotometer (U-2001, Hitachi Instruments Inc., Tokyo, Japan). A control contained all reagents with exception of extracts. Synthetic antioxidants butylated hydroxytoluene (BHT) was used as a positive control. Percent inhibition of linoleic acid oxidation was calculated with the following equation: 100 – [(increase in absorbance of sample at 360 h / increase in absorbance of control at 360 h) × 100], to express antioxidant activity.

Each extract was tested for its reducing power by FRAP assay with some modifications [25 (link)]. Briefly, 20 μL of the sample solution in DMSO with the concentration of 1 mg/mL was mixed with 180 μL of freshly prepared FRAP solution, which contains 0.3 M acetate buffer (pH 3.6), 10 mM 2,4,6 tripyridyl-s-triazine (TPTZ) solution in 40 mM HCl, and 20 mM ferric chloride (10:1:1), and kept in room temperature for 5 min. The absorbance was measured at 595 nm by using a multimode detector (Beckman Coulter DTX880, Fullerton, CA, USA). Ferrous sulfate (FeSO₄) was used as a standard and the ferric ions reducing power were expressed as equivalent capacity (EC₁) which represented the amount of FeSO₄ equivalents per mg of the sample. EC₁ was calculated using the following equation:

where a is an absorbance of sample solution with the present of FRAP solution and b is an absorbance of sample solution without the present of FRAP solution. All experiments were done in triplicate.

M. smegmatis cells expressing different M. tuberculosis proteins were grown in identical conditions to late log phase or stationary phase. In all expression cultures the ZYP–5052 autoinduction media was used for F₄₂₀ production experiments and the media to flask volume ratio was kept constant at 20%. In order to optimize the media for F₄₂₀ production, the ZY component of ZYM–5052 media was replaced by commonly used media bases including 2× ZY, YT (0.8% tryptone, 0.5% yeast extract and 42.77 mM NaCl), TB (1.2% tryptone, 2.4% yeast extract and 0.4% glycerol), SOB (2% tryptone, 0.5% yeast extract, 8.56 mM NaCl, 2.5 mM KCl and 10 mM MgCl₂) and SOC (SOB with 20 mM glucose). Iron and sulphur supplements (ferric ammonium citrate, ferric citrate and ferrous sulphate all at 0.1 mg/mL and L–cysteine at 1 mM) were also added to the expression media as a possible requirement for the FbiC enzyme. L–glutamate and manganese chloride (1 mM final concentration) were also added to the expression media to evaluate their necessity for FbiB–mediated F₄₂₀ production [22] (link).
To ascertain the optimum growth period for F₄₂₀ production, eight identical cultures of M. smegmatis cells expressing the recombinant FbiABC construct were set up. Each culture had a wild type M. smegmatis culture as a control. At 24 h intervals, one culture each of control and recombinant FbiABC–expressing M. smegmatis cells were harvested and processed to monitor the F₄₂₀ production level. The procedure was carried out for eight days and the F₄₂₀ production ratio for each day was calculated by dividing the F₄₂₀ fluorescence from FbiABC–expressing cells by fluorescence of the wild type control.
M. smegmatis cells were centrifuged for 15 min at 16000×g and the resulting media were used for FO characterization. The cell pellets were washed with 25 mM sodium phosphate buffer, pH 7.0 and were subsequently resuspended in 1 mL of the same buffer per 100 mg of cells (wet weight). The cell suspensions were autoclaved at 121°C for 15 min to break the cells open and were then centrifuged for 15 min at 16000×g. Fluorescence of the media and the extract were monitored using excitation wavelength of 420 nm (405±10 nm filter) and emission wavelength of 480 nm (485±15 nm filter). All fluorescence experiments were performed using an EnVision Multilabel plate reader (Perkin Elmer) in a 96–well plate format and were carried out in triplicate.
The autoclaved cell extracts were further purified using a HiTrap QFF ion exchange column (GE Healthcare) to separate the intracellular FO from the F₄₂₀. The extract was run on the column pre–equilibrated with 25 mM sodium phosphate buffer, pH 7.0 and was subsequently washed with five column volumes of buffer. Two yellow fractions were eluted at 200 and 500 mM NaCl, respectively. The purified fractions were used for mass spectrometry analysis, together with the media from the previous step. The media (1 mL) was treated with an equal volume of cold acetone to precipitate the protein and the solution was then evaporated down to <0.5 mL to drive off the acetone. A mix of water and 5% aqueous methanol with 0.1% formic acid was added to bring the final concentration of methanol to less than 1% (total volume 4 mL). All samples were then applied to a pre–equilibrated Alltech Maxi–Clean 300 mg large pore 100Å C–18 SPE cartridge and washed with 4 mL 5% methanol containing 0.1% formic acid followed by 4 mL 10% methanol. Compounds were eluted with 4 mL 80% methanol containing 5 mM ammonium bicarbonate pH 8.5. Eluates were evaporated under nitrogen and redissolved in 80% methanol and 20 mM ammonium acetate ready for mass spectrometry. Samples were infused at 3 µL/min under negative electrospray conditions into an LTQ–FT mass spectrometer (Thermo Scientific). The ion intensity data were obtained using a source voltage of 2.5 kV and capillary temperature of 225°C. Ions were examined in both the ion trap and ion cyclotron resonance cells, the latter to obtain high resolution (100,000 at m/z 400) accurate mass data. This was necessary to confirm the atomic composition of the ions and help deconvolute the contribution of metal ion adducts (Na⁺/K⁺) to the levels of individual poly–glutamate species. Up to four sodium ions were adducted to produce some double charged negative ions.

Ethanol, perfluoropentane, ferrous chloride tetrahydrate (FeCl₂·4H₂O) and ferric chloride hexahydrate (FeCl₃·6H₂O) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Epikuron^® 200 (soy phosphatidylcholine 95%) was a kind gift from Cargill (Wayzata, MN, USA). Palmitic acid was obtained from Fluka (Buchs, CH, Switzerland).