Materials S. reiliana was collected at the National Sorghum Improvement Center Base of the Liaoning Academy of Agricultural Sciences. The smut was harvested in mid-July. The bracts were removed, dried in the shade in a well-ventilated room, crushed, and the powder was stored until use. The extraction conditions of melanin has been reported by us previously (Lu and others, 2020): Ultrasonic power 245W, temperature 50°C, time 2 hr, and pH 13 of NaOH extract.
The chemicals and solvents used for extraction and analysis in this study were all purchased from Sigma-Aldrich (St. Louis, MO. USA) or Solarbio (Beijing Solarbio Science &Technology Co., Ltd, Beijing, China).
Solubility analysis One milligram of L-25 melanin was added to test tubes containing 2 ml of distilled water, HCl, dilute sulfuric acid, NaOH, ammonia, ethanol, methanol, ethyl acetate, dichloromethane, DMSO, acetone, or petroleum ether. The sample was mixed with each solvent and incubated for 3 h, after which the solubility of L-25 melanin was measured.
The effect of pH value on the stability of melanin L-25 melanin (0.5mg) was dissolved in ten milliliters of 0.1mol/L NaOH or HCl solution with pH values of 3.0, 5.0, 7.0, 9.16, and 10.83 were prepared. The color change of the solution was observed, and the absorbance value (A) was measured at 215nm.
Light stability A total of 1 mg of L-25 melanin was dissolved in 20 mL of 0.1mol/L NaOH solution, then divided it into 2 parts. One part was placed under natural light for 4 days and samples were taken every day. The second part was placed under UV light for 30 min or 60 min, and the absorbance value A was measured at 215nm.
Effect of microwaving on the stability of melanin L-25 melanin (50 mL) was prepared and heated in a microwave for different times (0-30 min). After cooling, the absorbance value A was measured at 215 nm.
Oxidation-reduction properties Various amounts of L-25 melanin solution were mixed with various volumes of vinyl chloride (VC), H2O2, Na2SO3, and sodium benzoate and added to 100 mL of water. After mixing, the solutions were placed in the dark for 1h. Subsequently, the light absorption value A was measured at 215 nm.
Metal ion stability A 0.5 mg sample of L-25 melanin was dissolved in 10 ml of a 0.1 mol/L NaOH solution containing 0.01 mol/L of metal ions (Ca2+, Fe2+, Zn2+, Na+, Al3+, K+, Mg2+, Fe3+, and Cu2+) to explore the effect of metal ions on the stability of L-25 melanin. Samples were taken every 12 h, and their absorbance was measured at 212 nm.
Detection of Melanin Complex with Cu2+ and Fe3+ by Infrared Spectroscopy A sample of L-25 melanin (25mg in 100 mL) was dissolved in 1 mol /L NaOH and filtered through a 0.45μm microporous filter membrane. The solution was evenly divided into two parts. One part was mixed with 25mL of a 0.02 mol/L CuSO4 solution. The other part was mixed with 25mL of a 0.02mol/L FeCl3 solution. Both mixtures were left for 12h. Next, the pH was adjusted to 2.0, 3.0, 4.0, or 4.5 using 1mol/L HCl, and the mixtures were left standing for 12h. The acidified solution was stewed, centrifuged, and the precipitate was dried under vacuum.
UV–Vis spectrum The L-25 melanin was dissolved in 0.1 mol/L NaOH at a final concentration of 0.05 mg/mL. The UV-visible absorption spectrum of the L-25 melanin was scanned in the wavelength range of 200~800 nm using a UV-visible spectrophotometer (Unico Instrument Co. Ltd.) with 0.1 mol/L NaOH as the reference.
Fourier-transform infrared spectroscopy The melanin samples were mixed with KBr (1:100 w/w) and homogenized. The mixture of melanin and KBr was pressed into a tablet and analyzed by Fourier-transform infrared spectroscopy (Bruker VERTEX 80) in the scanning range of 4000~400 cm-1.
Reversed-phase-HPLC preparation and separation conditions The melanin sample was analyzed by reversed-phase using a Chromatograph CX-3000. The detector was a CX-3000. The analytical column was a C18 (4.0×250mm). Mobile phase A was methanol and mobile phase B was water. The stepwise elution was as follows: 0 min 100% B, 20 min 30% A, 70% B, 50 min 100% A. The column temperature was 30 ℃. The flow rate was 14mL/min. The injection volume was 100μL.
Dynamic light scattering Dynamic light scattering (UPLC-QTOF-MS) analysis was performed using Shimadzu’s liquid chromatography system and a triple TOF 5600 mass spectrometer (AB Sciex) equipped with an electrospray interface. Liquid chromatography conditions were as follows: the chromatographic column was an SB-Aq C18 column (2.7μm), dimensions 3.0×100 mm (Agilent). The column was operated at 40°C. Pump A contained 0.05 mol/L ammonium formate, pump B contained acetonitrile. The elution flow rate was 1.0 mL/min. The elution was as follows: 0-8 min, 10-40% B; 8-14 min, 40-60%B; 14-18 min, 60-70%B; 19-26 min, 70-95% B. The data was collected at 520 nm. Melanin (1mg) was dissolved in 1 mL of methanol. Mass spectrometry analysis conditions were as follows: the injection volume of 1 μL was passed in the negative ion mode. The ionization source conditions were: atomization temperature 600℃, atomization voltage 4.5kV, DP -80, full scan spectrum in the range of 80-1500 m/z, cycle time was 0.2 s.
DPPH free radical scavenging ability measurement
Antioxidant activities of substances are mainly assessed by the determination of free radical scavenging ability using cell models and chemical methods, of which the 2,2-diphenyl-1-picrylhydrazyl (DPPH•) method is the most commonly used. The experimental method was as reported in Zheng et al. (2015). Briefly, two test solutions were prepared for sorghum black powder, melanin, and VC, with mass concentrations of 2 mg/mL and 0.4 mg/mL, respectively. DPPH powder (5.92 mg) was dissolved in absolute ethanol in a 250 mL brown volumetric flask. The solution (6×10-5 mol/L) was stored in a dark place until use. The DPPH free radical solution (2 mL) was added to different amounts of sorghum smut melanin sample solution (0.2, 0.4, 0.6, 0.8, and 1.0 mL) and each sample was brought to 4 mL with absolute ethanol. The samples were shaken well, and diluted with deionized water after 10 minutes. The absorbance of each solution was measured at 517 nm at different times. As a reference, the sample solution was replaced with an equal volume of deionized water, so that the detected system only contained DPPH radicals and absolute ethanol. In an additional control, the DPPH radical solution was replaced with an equal volume of water. The absorbance was measured using an ultraviolet spectrophotometer.
The calculation formula of the DPPH free radical scavenging rate was as follows:
where K represents the scavenging rate of free radicals; Ai represents the absorbance of the DPPH free radical solution after adding the sample to be tested; Aj represents the absorbance of the sample without DPPH radical solution; Ac represents the absorbance without adding the sample to be tested, i.e., only the absorbance of the DPPH radical solution.
The experiment was conducted as previously described 27 and the preparation of the different experimental solutions was as follows: An 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS)+·stock solution (7 mmol/L) was prepared by dissolving the ABTS free radicals in 2.45mmol/L potassium persulfate and stored away from light at room temperature for 12~16 h. This stock solution was stable for 3~4 d.
The ABTS+·determination solution was prepared by diluting the ABTS+· stock solution with absolute ethanol until its absorbance A reached 0.700±0.020 at the maximum wavelength of 734nm. Specifically, 40 μL of sorghum smut melanin sample test solution was added to 4 mL ABTS free radical test solution. The mixture was shaken for 30 s and the absorbance A was measured at the maximum absorption wavelength of 734 nm after various time intervals.
In vitro assays of cytotoxicity and proliferation
The effect of melanin on cytotoxicity and proliferation was measured using the thiazole blue (3-(4,5-dimethyl-2-thiazolyl)- 2,5-diphenyl tetrazolium bromide (MTT) method, which is widely used for cell viability studies (Cao et al. 2017). Specifically, growing HepG2 cells were selected for our viability studies. The cells (100 μL) were suspended in growth media and inoculated into 96-well plates (9000 cells per well). Following the addition of PBS (150 μL) to each well the cells were incubated in a CO2 incubator for 24 h to make the cells adhere to the wall. Subsequently, the prepared melanin samples from the sorghum smut fungus were added to each well of the 96-well plate according to the set concentration, placed into the incubator, and cultured for another 24 h. Then, the MTT solution (0.5mg/mL) was added to each well followed by a 4 h incubation. Then, the medium was aspirated and DMSO (150μL/well) was added, the plate was placed on a shaker and shaken well for 15 min, until complete color development. Finally, the 96-well plate was placed into a microplate reader and the absorbance at 490nm was read. In the experiment, the growth rate of HepG2 cells in the control group without melanin was recorded as 100% and the survival rate of HepG2 cells in the remaining groups was calculated according to the following formula:
Cell survival rate (%) = (OD experimental group-OD blank group)/(OD control group-OD blank group)×100%
The melanin-treated cells were washed with PBS and trypsinized using 0.25% Trypsin and 0.02% EDTA. The cell suspension was centrifuged for 5 min at 4000r/min, the cells were washed using PBS, and lysed with cell lysis buffer (150 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, 1 mmol/L EDTA, 1% TritonX-100,), followed by centrifugation at 4 ℃ for 1h. The above clear liquids were used as samples and intracellular ROS, MDA, and LDH content was determined using a kit according to the manufacturer’s instructions. Intracellular SOD and GSH-Px activities were determined using a kit according to the manufacturer’s instructions. The SOD activity unit was defined as one nitrite unit when the SOD inhibition rate reached 50% per milligram of tissue protein in a 1 mL reaction mix; the GSH-Px activity unit was defined as the reduction of non-enzymatic reactions per milligram of protein per minute, to reduce the GSH concentration in the reaction system by 1μmol/L. Protein quantification was determined by Coomassie Brilliant Blue colorimetry.
2.6 Statistical analysis
All experiments were repeated at least three times, and the results were expressed as mean ± standard deviation. The statistical significance was analyzed using SPSS 22.0 software, and P < 0.05 was considered statistically significant. Origin 7.5 software was used to produce graphs.