The pigments quantification was done with a UV-vis spectrophotometer (CLARIOstar, BMG LABTECH GmbH, Ortenberg, Germany). The total color value was assessed by measuring the absorbance of 75% ethanol extract of Hongqu at 505 nm. The result was expressed as the absorbance unit per gram at a specific wavelength (Srianta et al., 2016).

Determination of MK by HPLC was in accordance to the method in QB/T 2847-2007 and reference (Huang C. F. et al., 2019) with minor modification. Before chromatographic analysis, the supernatant was filtered through a 0.22 μm filter. The supernatant was measured by Waters e2695 with a chromatographic column (Zorbax SB-C18 column, 5 μm × 250 mm × 4.6 mm) and a UV detector at 238 nm. The mobile phase was composed of methanol:water:phosphoric acid in a ratio of 385:115:0.14 (v/v), with an accompanying flow rate of 1.0 ml/min.

γ-Aminobutyric acid was extracted and analyzed according to the method in QB/T 4587-2013 (Ministry of Industry and Information Technology of the People’s Republic of China, 2013). The content of GABA was determined by HPLC equipped with a reversed-phase column (Novapack C18, 300 mm × 3.9 mm × 4 μm, Waters, Milford, MA, United States). Eluted GABA was detected at 280 nm and expressed as mg/100 g dry Hongqu (Garzón et al., 2020).

The content of ergosterol was determined by HPLC equipped with an ODS18 column (250 mm × 4.6 mm × 5 μm, Waters, Milford, MA, United States) at 280 nm. Ergosterol in Hongqu was identified by a combination of the retention time in HPLC chromatograms with standards (Guan et al., 2016).

The preparation of Hongqu polysaccharides was under the method in the literature (Xie et al., 2012). The polysaccharide was detected by the phenol-sulfuric acid method (Dubois et al., 1956).

Determination of pigment compounds was in accordance to the method (Liang et al., 2019) with some modifications. The analysis was conducted by Agilent 2000 HPLC system (Agilent Technologies, Santa Clara, CA, United States) comprised of a binary pump, an autosampler, and a thermostatically controlled column compartment. Pigments were separated on a 250 × 4.6 mm ODS18 column (5 μm, Waters, Milford, MA, United States) with a linear gradient of the mobile phase of solvent A (water, containing 0.1% formic acid, v/v) and solvent B (acetonitrile), a flow rate of 1.0 ml/min, oven temperature of 35°C, and run time for 55 min. Mass spectrometry was operated with a 6520 QTOF-MS (Agilent Technologies, Santa Clara, CA, United States) equipped with an ESI source. The parameters of the ESI source were set as follows: capillary voltage, 3,500 V; fragmentor voltage, 135 V; drying gas (N2) temperature, 350°C; drying gas (N₂) flow rate, 10.0 L/min; nebulizer gas pressure, 30 psig; OCT RF V, 750 V; skimmer voltage, 65 V. Each sample was analyzed in positive modes with a mass scan range of m/z 50–1,200 Da, and collision energy (CE) was set as 15 and 30 eV. Data acquisition and analysis were operated by Agilent LC/MS Qualitative Analysis Software (version B.04.00).

The DPPH radical scavenging capacity was determined according to the method described by reference (Brand Williams et al., 1995) with minor modification. Each sample with a fixed volume of 1.0 ml was mixed with 1.0 ml DPPH solution (0.2 mM in anhydrous ethanol) stored in the dark at room temperature for 30 min. Then, its absorbance A_i was measured at 517 nm using a microplate reader (CLARIOstar, BMG LABTECH GmbH, Ortenberg, Germany). At the same time, the absorbance A_c of the mixture of DPPH solution and an equal volume of anhydrous ethanol and the absorbance A_j of the mixture of the sample solution and the equal volume of anhydrous ethanol were determined at 517 nm. The DPPH radical scavenging activity was expressed as the percentage of inhibition of DPPH according to the following formula. Inhibition rate of DPPH = [1- (A_i-A_j)/A_c] × 100%.

ABTS radical scavenging ability was determined according to the method with some modifications (Sridhar and Charles, 2019). The ABTS⋅⁺ working solution was produced by reacting the ABTS⋅⁺ stock solution (7.4 mM ABTS) with the K₂S₂O₈ stock solution (2.6 mM K₂S₂O₈) in equal quantities and allowed them to react for 12–16 h at room temperature in the dark and diluting with anhydrous ethanol to make its absorbance 0.70 ± 0.02 at 734 nm. ABTS⋅⁺ working solution (1.0 ml) was mixed with 0.25 ml of the ethanol extract of Hongqu or standard (dibutyl hydroxytoluene, BHT) at different concentrations (0.1–10 μg/ml). The mixture was incubated at room temperature for exactly 10 min in the dark and determined the absorbance at 734 nm (A). The control (A₀) was prepared by mixing 1.0 ml of ABTS⋅⁺ solution with 0.25 ml of anhydrous ethanol. The percentage results of scavenging activity were calculated as inhibition rate using the following equation. Inhibition rate of ABTS⋅⁺ = [(A₀ − A)/A₀] × 100%.

Ferrous-reducing power was determined as described in the literature (Eda et al., 2016) with some modifications. Sample solution (0.25 ml), phosphate buffer (0.25 ml) (pH 6.6, 0.2 mol/L), and potassium ferricyanide (0.25 ml) (10 g/L) were placed in a 1.5 ml centrifuge tube. After incubation at 50°C for 20 min and cooling to room temperature, 0.25 ml of 10% (m/v) trichloroacetic acid (TCA) solution was added and centrifuged at 4°C and 10,000 r/min for 15 min. Supernatant (0.5 ml), distilled water (0.5 ml), and 0.1% (m/v) ferric chloride solution (0.1 ml) were added and reacted for 10 min. The absorption was measured at 700 nm.

The superoxide anion was produced by the AP-TEMED system and reacted with hydroxylamine hydrochloride to form NO₂^–. Then, NO₂^– was reacted with paminobenzenesulfonic acid and α-naphthylamine to form a red azo compound with a characteristic absorption peak at 530 nm (Takei et al., 2017). The scavenging ability of oxygen anions was negatively correlated with the absorbance at 530 nm. O^2– radical-scavenging activity was measured by a non-enzymatic method with a test kit produced by Beijing Solarbio Science and Technology Co., Ltd.

The inhibition rate of yolk lipoprotein (AOA%) was determined as described in our previous report (Wu et al., 2016). Phosphoric acid buffer (3.0 ml) (0.1 mol/L), 1:25 yolk suspension (0.4 ml), FeSO₄ solution (0.2 ml) (25 mmol/L), and sample solution (0.2 ml) were placed in 10 ml tubes, respectively. The control tube (A₀) was placed with 0.2 ml of phosphoric acid buffer instead of the sample solution (A). After incubation at 37°C for 12 h, 1.0 ml of 20% trichloroacetic acid was added, centrifuging for 10 min at 3,500 r/min. Supernatant (4.0 ml) was reacted with 2.0 ml of 0.8% thiobarbituric acid solution for 15 min at 100°C. The absorbance was determined at 532 nm and calculated by the following formula, Yolk lipoprotein inhibition rate (AOA%) = [(A₀ − A)/A₀] × 100.

According to Figure 2, the units of absorbance at 505 nm (E₅₀₅) of CQ, indicating the total color value, were extremely significantly higher than those of FQ. The average value of E₅₀₅ of CQ was approximately 2,569.60 U/g, while that of FQ was only 283.24 U/g. The E₅₀₅ of CQ was approximately 9.0 times compared with that of FQ. For example, Huang et al. (2017) reported a higher antioxidative potential in a fermented mixture of Radix Puerariae and rice than in that fermented by only Radix Puerariae and rice due to the higher levels of pigments. The total color value of red yeast rice (RYR) and fermented Radix Puerariae and rice (FPR) was 1,300 ± 25 units/g and 6,164 ± 799 units/g, respectively. FPR increased the color value by three times compared with RYR. The result indicated that the total color value between CQ and FQ was different, so the differences in Monascus pigments composition needed to be further analyzed.

Monacolin K is one of the well-documented metabolites of Monascus and is recognized as a cholesterol-lowering regent because of its competitive inhibitory effect on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. The total amount of MK is composed of the lactone form and the acid form of MK. The content of MK in Hongqu was shown in Table 2. The total contents of MK of FQ 1–FQ 5 were 33.73, 20.27, 6.98, 9.02, and 10.66 mg/g, respectively. So, FQ 1–FQ 5 met the requirements of the light industry standard of the People’s Republic of China that the MK of FQ should be over 4.0 mg/g. The ratios of the active open-hydroxyl acid form and the prodrug lactone form of MK of FQ 1- FQ 5 were 13.64, 12.88, 26.93, 18.18, and 27.02%, respectively. The MK of CQ 4 was the highest among all CQ samples, reaching 0.26 mg/g (less than the requirements of FQ). Gum et al. (2017) reported higher antioxidant activity in Hongqu fermented Bacillus subtilis than in Hongqu due to the alteration in the physicochemical property of MK.

The contents of GABA, polysaccharide, and ergosterol of CQ were 0.103, 14.58, and 246.92 mg/g, while those of FQ were 0.198, 35.97, and 443.65 mg/g, representing increases of 92.23, 146.70, and 79.67%, respectively. The contents of GABA, polysaccharide, and ergosterol of FQ were higher than those of CQ. However, there was no significant difference between them (Figure 3). Lee et al. (2016) reported that amino acids contributed to antioxidant activity in wheat and rice gochujang. Lu et al. (2015) reported that Huangjiu had the highest antioxidant capacity among the three traditional fermented wines (Baijiu, Huangjiu, grape wine) because the amino acid content was 2,923 μg/ml, and GABA was 10 μg/ml. Numerous studies reported that polysaccharides from different sources and modified products had certain antioxidant activities (Mirzadeh et al., 2020). Liang et al. (2019) reported that the ergosterol in RYR could significantly reduce the levels of total cholesterol, triglycerides, and low-density lipoprotein cholesterol in C57BL/6J mice fed a high-fat diet. Therefore, GABA, polysaccharides, and ergosterol might be potential contributors to antioxidant activity.

The antioxidant activities of seven MYPs were evaluated by antioxidant assay in vitro, including the DPPH free radical scavenging rate (Figure 7A), superoxide anion clearance rate (Figure 7B), and inhibition rate of peroxidation of yolk lipoprotein (Figure 7C). The three chemical assays reveal the different antioxidant characteristics of MYPs based on different antioxidant mechanisms. The antioxidant capacity of MYPs increased with the increase of concentration. The antioxidant activities of MYP were similar in the three chemical antioxidant assays. Chemical antioxidant tests revealed that the antioxidant activities of monaphilone A, ankaflavin, and new yellow pigment only separated from CQ were significantly stronger than monasfluore A and monascuspilion only separated from FQ. For example, the IC₅₀ of the new yellow pigment in three chemical assays (0.062, 0.376, and 0.580 mg/ml) was smaller than that of monascuspiloin (0.080, 0.548, and 2.670 mg/ml). This might be the main reason for the significantly stronger antioxidant capacity of the crude ethanol extract of CQ than FQ. MYPs have good DPPH radical scavenging ability and superoxide anion scavenging capacity, and bad vitellin inhibitory capacity. The IC₅₀ of the DPPH free radical scavenging rate of MYPs from FQ (0.080–0.091 mg/ml) was smaller than the IC₅₀ of crude ethanol extracts of FQ (0.544 mg/ml). The results indicated that the antioxidant activity of yellow pigments was improved significantly after purification.

Compared with the antioxidant assay in vitro and animal experiments, the evaluation of antioxidant activity by cell model test was more economical and faster. It is widely used to quantify the cellular antioxidant activity (CAA) of phytochemicals, food extracts, and nutritional supplements. Therefore, we consider CAA assays as reliable and sensitive methods for antioxidative activity evaluation. With quercetin as a positive control, CAA of seven pigments was evaluated with an oxidative damage model of human colon adenocarcinoma Caco-2 cells induced by AAPH (Kellett et al., 2018). The CAA value of MYPs at the 20 μg/ml concentration showed that the new yellow pigment (65.46 units) had the best antioxidant activity, followed by monasfluore A (42.15 units) and monaphilone B (39.19 units), which were all significantly better than monascin (26.86 units) and ankaflavin (22.19 units), the two most frequently reported MYPs (Figure 8A). The antioxidant capacity of the natural product was usually converted to an equivalent concentration of quercetin based on its CAA values for making the result more comparable (Zhou et al., 2020). The quercetin equivalent of MYPs was shown in Figure 8B. The quercetin equivalent of the new yellow pigment was 7.23 μM. In short, the new yellow pigment had outstanding antioxidant capacity in vitro, and its antioxidant capacity in vivo was worthy of in-depth study.

With the help of in vitro and in vivo antioxidant experiments, the relationship between the structure and antioxidant activity was further investigated. Based on the structure of 111 identified single pigment, the tricyclic carbon skeleton structure or the dicyclic carbon skeleton was the core structure of MonAzPs, which generally contained one to three carbonyl groups and two to five unsaturated double bonds. To date, a unitary trunk pathway had been revealed to produce four classical pigments and intermediates. All other 106 species MonAzPs were generated by various shunt pathways branching off from the trunk pathway (Chen et al., 2019). First, the alterations in the position and number of hydroxyl groups affect the antioxidant activity. The hydroxyl group of C-4 was prone to condensation reaction to form a lactone ring C5-C2′ condensation and cyclization formed a C5-C2′ linear tricyclic carbon skeleton or C3-C2′ condensation and cyclization formed a C3-C2′ angle tricyclic carbon skeleton. The seven yellow pigments could be divided into three categories according to carbon skeleton characteristics and the CAA. New yellow pigment and monasfluore A (Figures 9F,G) had an angular tricyclic carbon skeleton, obviously different from the linear tricyclic core of classical monascin, ankaflavin, and monascuspilion (Figures 9A,B,E). It is worth noting that the antioxidant activity of the new yellow pigment and monasfluore A was stronger and that of monascin, ankaflavin, and monascuspilion. Monaphilone A and monaphilone B had moderate antioxidant activity lacking the γ-lactone ring (instead of a C-4 hydroxyl group) (Figures 9C,D). First of all, alterations of the hydroxyl group of MYPs affect the antioxidant activities significantly. The results showed that the new yellow pigment with a hydroxyl group on C-11 possessed much higher antioxidant activities than monasfluore A and the others with a C10 (11) double bond on C-11. Monascuspilion with a hydroxyl group on C-3′ brought higher antioxidant activities than monascin with a ketone group on C-3′. In the case of MYPs, alterations of the γ-lactone ring and alkyl side chain could affect the antioxidant activities slightly. Monaphilone A and monaphilone B (lacking γ-lactone ring) possessed higher antioxidant activity than ankaflavin and monascin (linear tricyclic core), respectively. Moreover, monaphilone B and monascin (R = C₅H₁₁) had higher antioxidant activities than monaphilone A and ankaflavin (R = C₇H₁₅). In short, the alterations that occurred at C-3′, C-11, and the carbon skeleton more obviously affected the antioxidant activities.

The inhibitory effects of azaphilonoid pigments on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema were much stronger than those of non-azaphilonoid pigments (Akihisa et al., 2005). The critical structures that enhance the anti-inflammatory activity of Monascus yellow and orange pigments were found by Hsu (Hsu L. C. et al., 2013), and the structure–activity relationship was consistent with the results of the relationship between the structure and antioxidant activity of MYPs in this article. Because inflammation is a kind of exogenous stress, the Keap1-Nrf2/ARE antioxidant signal pathway is often activated (Lee B. H. et al., 2013). Nrf2 is an important transcription factor that regulates cell protection mechanisms such as antioxidant and anti-inflammatory activities (Pall and Levine, 2015). Therefore, anti-inflammatory activity is closely related to antioxidant activity. It is worth noting that MonAzPs with hydroxyl groups on C-3′, C-11 had significantly stronger antioxidant activity than a ketone group and C10 (11) double bond on C-3′, C-11, respectively. The previous studies had reported that the antioxidant potential of MYPs could easily transfer hydrogen to and accept electrons from free radicals through HAT and SET mechanisms. This result was consistent with the alterations of key antioxidant structures of MYPs.