Characterization of the physicochemical properties, antioxidant activity, and antiproliferative activity of natural melanin from S. reiliana

This study aimed to characterize the physicochemical properties and stability of L-25 melanin extracted from Sporisorium reilianum (S. reiliana). The results showed that the maximum absorption wavelength of melanin was 215 nm. Reducing agents, heat, light, microwaving, oxidants, and common food additives did not affect the melanin. Additionally, it has a good metal stability except Mn2+. The IR spectra revealed the presence of O–H, N–H, C=O, and C=C bonds as well as carboxyl, alcohol hydroxyl, and phenolic hydroxyl groups and a pyran ring. L-25 melanin could be defined as DL-hydroxy phenylalanine (DOPA)-melanin. The antioxidant and antiproliferative were also measured. The melanin has a specific stability and high antioxidant activity, including a strong DPPH free radical scavenging ability, and protected damaged HepG2 cells by reducing reactive oxygen species, malondialdehyde, and lactate dehydrogenase content. In conclusion, S. reilianum represents a novel source of melanin, that could be applied to health food or food additives. Our results show that melanin from S. reilianum is a natural pigment with good stability that has a great prospect of development and application, providing a theoretical basis and methods for its further processing and development as a functional food.


Scientific Reports
| (2022) 12:2110 | https://doi.org/10.1038/s41598-022-05676-z www.nature.com/scientificreports/ are commonly used in the private sector. It has many effects, such as regulating menstruation, hemostasis, lowering blood cholesterol, weight control, and preventing colitis and coronary heart disease. However, the research focus on S. reiliana is mainly as a plant pathogen infection mechanism and classification. In recent years, active metabolites of S. reiliana have been studied extensively, and multiple new active compounds have been identified 2 .
Although melanin is an important active ingredient in S. reiliana, its active fractions and chemical structures related to antioxidative effects have rarely been addressed.
In this paper, we studied the physicochemical properties of L-25 melanin that was extracted from S. reiliana as previously reported 3 . The melanin was analyzed by scanning electron microscopy (SEM), dynamic light scattering (ultra-performance liquid chromatography (UPLC)-quadrupole time of flight mass spectrometry (QTOF-MS)), elemental analysis, ultraviolet-visible spectrometry (UV-Vis), and infrared spectrometry (IR). Furthermore, the antioxidative effects and antiproliferative activities of the purified melanin were studied. Our results provide insights into the physicochemical properties of melanin extracted from S. reiliana and its potential use in functional foods or food additives.

Results and discussion
Physicochemical properties of L-25 melanin. As shown in supplementary materials (Table S1), L-25 melanin had relatively low solubility in water, acid, and most organic solvents including ethanol, dichloromethane, ethyl acetate, acetonitrile, and acetone. However, in strong alkaline solutions, such as NaOH, the solubility was higher. The melanin was slightly soluble in dimethyl sulfoxide and methanol. It can precipitate out in a strong acidic solution. This solubility characteristic of L-25 melanin was very similar to melanin obtained from other microorganisms.
The effects of the environmental conditions including illumination, temperature, microwaves, metal ions, oxidizers, and reducers on the stability of L-25 melanin were also studied. The pH value strongly influences the melanin of sorghum black powder fungus, including changes in light absorption value and color (Table S2). The melanin appeared brownish red in the range of pH 2-7 and turned dark brown when pH > 8. The effect of light on melanin was not obvious, but with prolonged exposure to natural light or ultraviolet light irradiation, there was a slight increase in the light absorption value (Table S3). The reason may be the enhancement of the polymerization of melanin from sorghum during exposure to light. We also speculated that L-25 melanin has a certain protective effect against ultraviolet radiation. With the extension of microwave time from 0 to 30 min, the absorbance of melanin hardly changed, indicating that microwave treatment had little effect on L-25 melanin (Table S4). Our study also found that treatment with the reducing agent (Na 2 SO 3 , VC and sodium benzoate) had no significant effect on the stability of L-25 melanin. Moreover, the reducing agent Na 2 SO 3 had no significant effect on the stability of L-25 melanin. However, the oxidizing agent H 2 O 2 reduced the stability of IH melanin (Tables S5-S7).
Among the metal ions used, Mn 2+ had the most significant impact on the stability of L-25 melanin (Table 1), which may be related to complex reactions between the metal ions and several neighboring phenolic hydroxyl groups of melanin 9,17 . The remaining metal ions had no significant effect on the stability of the melanin compared with the control. Cu 2+ and Fe 3+ play a very important role in the formation of melanin. The active site of tyrosinase, a key enzyme in the process of eumelanin formation, contains both a Cu 2+ and Fe 3+ ion. The infrared spectrum characteristics of complexes between metal ions Cu 2+ , and Fe 3+ and L-25 melanin were compared to that of sorghum melanin not complexed to a metal ion (Fig. 1). We found that the pH value of the complex and the solution affected the complexation of melanin and metal ions. The effect on the complex sum of the two ions is similar. At pH 2.0-4.5, metal ions interact primarily with carbonyl groups. With the increase of pH value, metal ions interacted with L-25 melanin.
UV-visible absorption spectra. The maximum absorption of melanin was at 210 nm and decreased towards the visible region, which is a characteristic property of melanin (Fig. 2). This is because there are complex conjugated structures in the melanin molecules 19 . The decrease in absorption of L-25 melanin with the increase in wavelength is almost linear, and the log of absorbance against wavelength produces a linear curve with a negative slope of − 0.0031, which is one of the characteristics of melanin. Table 1. The effect of different metal ions on the stability of melanin. *Differences are significant (P < 0.05). **Differences are highly significant (P < 0.01).   Dynamic light scattering. L-25 melanin was prepared by RP-HPLC and the mass after concentration was about 20 mg. Melanin is an indole polymer and belongs to the group of eumelanin pigments. Studies have shown that eumelanin is derived from the black-brown insoluble melanin pigment subgroup 20 , which is derived from L-DOPA through the oxidative polymerization of 5,6-DL-hydroxy indole intermediates 13 . When the maximum ultraviolet absorption wavelength was 215 nm, the dynamic light scattering spectrum showed that L-25 melanin had 7 main peaks (Fig. 4). After baseline separation by UPLC, the 7 component peaks were characterized by monitoring molecular ion characteristics. The mass spectrum results, the m/z ratio of the molecular ion peak and the ion fragments produced, and the QTOF-MS ion diagram are shown in supplementary materials (Table S9).

Antioxidant activities.
The relatively stable organic radical DPPH has been widely used for the determination of the antioxidant activity of antioxidant compounds 21 . DPPH-containing solutions in ethanol appear purple in ethanol and have a strong absorbance at 517 nm. When antioxidants are paired with DPPH single electrons a gradual reduction of the color of the solution occurs 22 . The DPPH scavenging activities of L-25 melanin increased with increasing concentrations (Fig. 5) and showed a dose-response relationship. When the concen-  www.nature.com/scientificreports/ tration of melanin was 0.5 mg/mL, the DPPH scavenging rates of L-25 melanin reached 72.35%. The activity of DPPH free radical scavenging by L-25 melanin was remarkably higher than that of melanin from black sesame (47.7%) 23 . Hydroxyl radicals readily react with biological molecules, such as amino acids, DNA, and proteins,   24 . Therefore, it is necessary to find safe and effective antioxidants that scavenge hydroxyl radicals. The hydroxyl radical scavenging activities of L-25 melanin increased with increasing concentrations and exhibited a dose-response relationship. At a concentration of 0.4 mg/mL, the hydroxyl radical scavenging rate of L-25 melanin reached 76.86%. However, the ability of VC to scavenge DPPH free radicals was much greater than that of L-25 melanin. At the same time, a comprehensive comparison indicated that the DPPH free radical scavenging rate of VC was faster than that of L-25 melanin. In summary, the melanin from sorghum smut had the ability to remove DPPH free radicals but displayed only 20% of the removal activity of VC.
The ABTS radical cation is another commonly used organic radical to determine the antioxidant activity of single compounds or complex mixtures 25 . As shown in Fig. 5c, the effect of melanin and Vc on the ABTS free radical scavenging capacity was not significant with the extension of reaction time, the reaction rate of these two samples with ABTS free radical was fast and quickly reached equilibrium. The maximum clearance rate of melanin was 76.13%. This indicated that it was effective in scavenging ABTS free radicals.

L25 melanin protects HepG2 cells against H 2 O 2 -induced oxidative damage. Cell proliferation is
an important feature of life. It refers to the process of cell division through reactions such as DNA replication, RNA transcription, and protein synthesis. The growth of cancer cells can be evaluated by calculating the number of dividing cancer cells or the changes in the cell population, and this allows the determination of the effect of growth stimulation or inhibition of a sample 26 . This technology is widely used in many research fields such as tumor biology, molecular biology, pharmacokinetics, and pharmacology. It is not only critical for the study of the basic biological characteristics of cells, but also a basic method for analyzing cell states and studying genetic characteristics. The method can provide valuable information for exploring the pathogenesis, diagnosis, and treatment of a disease. Herein, the HepG2 cell line was used to evaluate the cytotoxic effect of L-25 melanin. As shown in Table 2, the survival rate of each mass concentration group is above 90%, and it had a certain proliferation effect at 200 mg/L. The results showed that melanin from sorghum smut had no obvious toxic effect on HepG2 cells in the concentration range of 10-400 mg/L (P < 0.05). Next, the effect of melanin at different concentrations and different times on the survival rate of HepG2 cells that were exposed to H 2 O 2 was determined ( Table 3). Compared with the control group, the cell survival rate of the H 2 O 2 injury group had significantly decreased (P < 0.05). Compared with the H 2 O 2 injury group, the cell survival rate of the sorghum smut melanin intervention group at a concentration of 10-100 mg/L was lower than that of the H 2 O 2 injury group. However, when the sample concentration was 200 mg/L, the 6 h, 12 h, and 24 h treatments all showed significant protection (P < 0.05). The protective effect was greatest when the concentration of melanin reached 400 mg/L, and the survival rate reached 85%, which was 15% higher than the survival rate in the H 2 O 2 injury model group.  As shown in Table 4, the values of ROS, MDA, and LDH in the H 2 O 2 model group were 364.529 ± 4.106, 5.071 ± 0.262, and 5691.393 ± 22.896, respectively, and these values were significantly higher than those of the control group (P < 0.05). Compared with the model group, the three intervention groups at high, medium, and low concentrations of sorghum smut melanin displayed significantly reduced levels of ROS, MDA, and LDH in HepG2 cells that are damaged by hydrogen peroxide, and this effect was concentration dependent. It is inferred from these results that sorghum smut melanin can protect damaged HepG2 cells by reducing the levels of ROS, MDA, and LDH. Compared with the control group, the activity of SOD and GSH-Px in the H 2 O 2 injury model group decreased significantly (P < 0.05) ( Table 5). Compared with the H 2 O 2 injury model group, the three intervention groups, treated with (high, medium, and low concentrations of sorghum smut melanin, respectively, displayed enhanced activities of the endogenous antioxidant enzymes SOD and GSH-Px, which confirms that sorghum smut melanin can protect HepG2 cells injury induced by H 2 O 2 . These results confirm the protective effect of melanin against cellular oxidative damage and its antioxidant activity.

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 245 W, temperature 50 °C, time 2 h, 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 and 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 (0.5 mg) was dissolved in ten milli- 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.  Fourier-transform infrared spectroscopy. The melanin samples were mixed with KBr (1:100 w/w) and

Detection of melanin complex with Cu
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 × 250 mm). 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 °C. The flow rate was 14 mL/min. The injection volume was 100 μL.

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. 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 min. 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. Distilled water was used as a control, V c was used as positive control. 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; A i represents the absorbance of the DPPH free radical solution after adding the sample to be tested; A j represents the absorbance of the sample without DPPH radical solution; A c 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 www.nature.com/scientificreports/ (7 mmol/L) was prepared by dissolving the ABTS free radicals in 2.45 mmol/L potassium persulfate and stored away from light at room temperature for 12-16 h. This stock solution was stable for 3-4 days. 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 734 nm. 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. Distilled water was used as a control, V c was used as positive control.
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 CO 2 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 (0.5 mg/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 490 nm 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 4000 r/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 °C for 1 h. 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 1 μmol/L. Protein quantification was determined by Coomassie Brilliant Blue colorimetry.

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.

Conclusion
In this work, we systematically studied the physicochemical properties and the in vitro antioxidant and antiproliferative activity of L-25 melanin extracted from S. reiliana. The results showed that the L-25 melanin can be defined as DOPA-melanin with a maximum UV absorption at 215 nm. L-25 melanin has a good thermal stability and light resistance, good solubility under alkaline conditions, and L-25 melanin was stable in the presence of most metal ions. It also had significant antioxidant activity. The results on antioxidant and antiproliferative activities have shown that melanin of Sorghum has moderate antioxidant activity in melanoma cells.
Our cytotoxicity experiments showed that the melanin of S. reiliana protected HepG2 cells against H 2 O 2 -induced damage. The melanin can protect the damaged HepG2 cells by reducing the ROS, MDA, and LDH contents. In addition, compared with the H 2 O 2 injury model group, three intervention groups, with high, medium, or low concentrations of melanin could enhance the activities of endogenous antioxidant enzymes SOD and GSH-Px, which confirmed the protective effect and antioxidant activity of melanin from S. reiliana against H 2 O 2 -induced oxidative damage in HepG2 cells. In the future, additional studies will be carried out on L-25 melanin to clarify its distribution and metabolism in vivo, and its applications in food and biomedical fields.