A black rice-derived mixture of anthocyanins with a purity >25% (mainly containing Cyanidin-3-glucoside) was obtained from Xi 'an Xiaocao Plant Technology Co., Ltd. (Xi 'an, China). Hyaluronic acid (HA) (purity >95%) was purchased from Henan Sanhua Biological Technology Co., Ltd. (China). Xanthine oxidase (Cat. NO. X1875, from bovine milk, ≥0.4 units/mg protein, 5U) and xanthine (purity ≥99%) were purchased from Sigma–Aldrich Co. Ltd. (St. Louis, USA). Other chemicals were all analytical grade and were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All experimental water was deionized water.

The HA/ATC mass ratio (8–12: 1), reaction pH (2.8–4.8), and stirring time (1–5 h) were considered as the experimental factors for the preparation of HAA nanocomposite particles. The nanocomposite particles were prepared according to a previously reported method (22) with slight modifications. HA (18 mg) was dissolved in 10 mL of deionized water and stirred continuously until a clear and transparent solution was obtained. Then, under the action of magnetic stirring, 2 mL black rice ATC solution was slowly added into HA solution with a syringe and mixed evenly. The pH was adjusted to 4.3 with dilute hydrochloric acid (1.0 M). After ultrasonic treatment for 5 min (power 200 W, frequency 59 kHz, working for 10 s, intermittent 5 s), magnetic stirring was performed for 3 h to obtain a uniform pink suspension. Finally, the suspension was dried in a vacuum freeze to obtain HAA nanocomposite particles. All the above experiments were carried out in a dark environment.

Black rice ATC's EE (%) was determined using the methods described previously (14, 23). The nanocomposite particles were centrifuged at 8,000 rpm for 30 min, and the free black rice ATC content in the supernatant was determined by the pH differential method. The samples were diluted with pH 1.0 (0.025 M) and pH 4.5 (0.4 M) buffers, and the absorbance was determined with distilled water as a blank at 510 and 700 nm, respectively. The black rice ATC content was calculated by the equivalent of cyanidin-3-glucoside (C3G) according to the equation (1): (1)c = (ApH1.0-ApH4.5) × Mw × DF × 1000Ma × L where ApH1.0 and ApH4.5 are the maximum absorbance of pH 1.0 and pH 4.5 buffer dilution samples, respectively. Mw is the molecular equivalent (449.2 g/mol) of C3G. DF is the dilution factor; Ma is the extinction coefficient (26,900 mol/L^*cm); L is the optical diameter (1 cm); and 1,000 is the conversion factor.

The encapsulation efficiency of the black rice ATC was calculated according to the equation (2): (2)EE(%)=ATC0-ATCtATC0×100 where ATC0 is the initial content of black rice ATC in the nanocomposite particles, and ATCt is the content of free black rice ATC in the supernatant.

The particle size, zeta potential, and polydispersity index (PDI) of HAA nanocomposite particles were analyzed using a Malven Zetasizer Nano-ZS (Nano-ZS) (Malvern Inst. Ltd., UK) at 25°C. Scanning electron microscopy (SEM) (SU8010, Hitachi Ltd., Japan) and transmission electron microscopy (TEM) (H-7650, Hitachi, Tokyo, Japan) were used to determine the surface morphology of the samples. X-ray diffractometry (XRD) (D8 Advance, Brucker, Karlsruhe, Germany) was used to scan and determine the structure and composition of the nanocomposite particles at room temperature. The FT-IR spectra of nanocomposite particles were determined by Fourier Transform Infrared Spectrometer (FT-IR) (IS50, USA) scanning in the range from 4,000 cm⁻¹ to 400 cm⁻¹.

The nanocomposite particles were stored for 6 days at 25°C and protected from light under 0.1, 0.25, 0.5, and 1.0 mg/mL ascorbic acid (AA) conditions. Samples were taken every day to detect the retention rate of black rice ATC. Sampling and detecting the retention rate of black rice ATC at appropriate intervals at three conventional temperatures of 4°C (cooling temperature), 25°C (room temperature), and 40°C (accelerated heating) (13). The nanocomposite particles were exposed to light and stored at 25°C for 10 days, and the retention rate of black rice ATC was measured every other day. The retention rate of black rice ATC was calculated by the pH differential method according to the equation (3, 24): (3)R(%) = CtC0×100 where R represents the black rice ATC retention rate (%) (defined as the percentage of ATC content change); Ct represents the black rice ATC content sampled at time t (mg/L); and C0 represents the initial black rice ATC content (mg/L).

The in vitro sustained release analysis of nanocomposite particles referred to a slightly modified version of the previous method (25). The lyophilized nanocomposite particle powder was suspended in phosphate-buffered saline (PBS) buffer (0.1 M, pH 7.4) to fully dissolve it, transferred to an 8–14 kDa dialysis bag, sealed and put into a vial containing 50 mL PBS buffer, and shaken at 100 rpm (37°C). Then, 3 mL of the fluid outside the dialysis bag was removed at a fixed point, and fresh buffer solution with the same temperature and amount was immediately added. The drug release amount and cumulative drug release percentage were calculated, and the sustained release curves of the nanocomposite particles were plotted in vitro.

The in vitro simulated digestion of nanocomposite particles was evaluated according to a previous method (26) with slight modifications. Simulated oral digestion: 1 mL of activated saliva (6.5 mg α-amylase and 0.5 mg CaCl₂ dissolved in ultra-pure water at pH 6.75) was added to a conical flask containing a 10 mL sample and digested at 100 rpm for 10 min (37°C). Simulated gastric digestion: After 10 min of simulated oral digestion according to the above steps, the oral digestive juice was adjusted to pH 2.0 with 6 M HCl, and then 1 mL of activated gastric juice (0.3 g of pepsin dissolved in 0.1 M HCl at pH 2.0), mixed well, and then shaken for 2 h. Simulated intestinal digestion: After oral and gastric simulated digestion according to the above steps, the gastric digestive juice was adjusted to pH 7.5 with 0.9 M NaHCO₃, and then 10 mL of activated intestinal juice was added (3.8 g of pig bile salt and 0.6 g of trypsin were dissolved in 0.1 M NaHCO₃ at pH 7.5) and mixed well, followed by shaking for 2 h. The retention rate of black rice ATC at different stages of simulated digestion in vitro was determined according to the method described in section stability analysis.

To compare and analyze the inhibitory activities of nanocomposite particles on XO in vitro, the inhibition experiment was carried out according to a previous report with slight modifications (27). Briefly, a series of mixtures consisting of a standard concentration of XO (0.012 U/mL), PBS buffer (0.05 M, pH 7.5), and the samples were incubated in a 25°C constant temperature water bath for 30 min. Then, 1 mL xanthine (0.2 mg/mL) was added to start the reaction. Then, the time/kinetics software UV–Visible spectrophotometer (U3010, Hitachi Ltd., Japan) was used to determine the absorbance of uric acid at 290 nm in the reaction system and to determine the catalytic activity of uric acid produced by XO in the presence of nanocomposite particles with different concentrations. Then, the relative inhibition rate of the sample to XO was calculated according to the equation (4): (4)I(%) = (1-BA)×100 where B represents the enzyme reaction activity in the presence of inhibitor, and A represents the enzyme reaction activity in the absence of inhibitor.

All data are expressed as the means ± standard deviations of triplicate determinations. One-way analysis of variance (ANOVA) and Duncan's test (P < 0.05) were performed by SPSS 26.0 (IBM Inc., Armonk, NY, USA) to compare the differences between groups. Origin 8.0 (Origin Lab Inc., Massachusetts, USA) was used to draw relevant charts.

As shown in Figure 2, the diffraction peaks of black rice ATC were mostly sharp, and characteristic peaks were observed at 2θ values of 15.47°, 23.36°, 31.71°, and 45.55°, which indicated that black rice ATC was highly crystalline. Two relatively wide diffraction peaks were detected at 2θ values of 11.31° and 19.73°, indicating that HA was semicrystalline. The diffraction peak intensity of the synthesized nanocomposite particles was significantly reduced, showing the characteristics of amorphous polymers. The results showed that the crystal structure of the black rice ATC molecule in the nanocomposite particles was covered, and that the diffraction peak intensity was significantly reduced, which may be related to the newly formed intermolecular hydrogen bond between black rice ATC and HA.

The averaged and smoothed FT-IR spectra of HA, black rice ATC, and HAA nanocomposite particles are shown in Figure 3, and the characteristic peak positions of HA, black rice ATC and HAA are shown in Supplementary Table 6 (30, 31). The positions of the characteristic peaks were determined according to previously methods (23, 32, 33). The peaks of black rice ATC at 1,641 and 1,444 cm⁻¹ corresponded to the stretching vibrations of C=C and C=O in the aromatic ring skeleton, respectively. A band at 1,326 cm⁻¹ corresponded to C-O angular deformations of phenols (13), and 1,245 cm⁻¹ was the stretching vibration peak caused by the benzopyran aromatic ring, which was a typical flavonoid structure peak. The peaks of HA at 1,633 and 1,417 cm⁻¹ belonged to the -COO⁻ group of carboxylic acid salt. In the spectral bands of the synthesized nanocomposite particles, the stretching vibration of C=O (1,648 cm⁻¹) showed a blueshift absorption peak, and the stronger stretching vibration at 1,303 cm⁻¹ was attributed to the introduction of black rice ATC, while the absorption peak at 1,046 cm⁻¹ was stronger than that of HA, indicating hydrogen bond formation. Moreover, there was a shift in O-H stretching vibrations from 3,382 to 3,449 cm⁻¹, revealing hydrogen bonding between black rice ATC and HA (34). The characteristic peak of ATC disappeared at 1,245 cm⁻¹ in HAA, indicating that HA had successfully embedded black rice ATC. In addition, the COO⁻ characteristic peak of HA at 1,417 cm⁻¹ was shifted to 1,409 cm⁻¹ in HAA, indicating the electrostatic interaction between -COOH of HA and -OH of black rice ATC. The above results all confirmed the successful combination of HA and black rice ATC.

Affected by its own structure, ATC are often unstable to the external environment and are easily degraded. The expectation that HA would stabilize black rice ATC by providing a barrier is supported by studies examining free black rice ATC and HAA storage. The stability analysis of free black rice ATC and HAA nanocomposite particles under different environmental stresses and storage conditions is shown below.

Light is an important factor affecting the degradation of ATC. As shown in Figure 6A, after 10 days of light treatment, the retention rate of free black rice ATC was 23.28%, while the retention rate of HAA nanocomposite particles increased by 43.63%, showing an obvious protective effect. ATC are prone to discoloration and degradation during exposure to light (39). As shown in Figure 6B, with the prolongation of light, the color of free black rice ATC changed from pink to light yellow and finally to dark yellow. In contrast, the color change of HAA nanoparticles was less obvious, indicating that HAA effectively protected the degradation of black rice ATC under light and improved its stability. The color changes of free black rice ATC and HAA nanocomposite particles were consistent with the change trend of black rice ATC retention rate, which indicated that HA embedding can effectively reduce the degradation of black rice ATC and improve the light stability.

The above conclusion could be explained from the strong interaction between HA and black rice ATC observed in XRD and FT-IR spectra. We believe that the stability of the HAA nanocomposite particles is due to the high permeability barrier of the HA carrier in the outer layer to polar oxidants (such as ascorbic acid) and degradation under heat and light.

XO is a key enzyme that produces uric acid in the human body and is a common drug target. Inhibiting XO activity becomes the main way to reduce the production of uric acid. ATC has a strong inhibitory effect on XO activity in vitro (10). To further test whether the synthesized HAA nanocomposite particles improved the inhibitory effect on XO activity, the activity of producing uric acid catalyzed by XO in the reaction system was detected in vitro. According to the mass ratio of HA and black rice ATC of 9:1, the inhibitory effects on XO activity were detected, and the results are shown in Figure 9. The in vitro inhibitory rate of black rice ATC (0.2 mg/mL) was 16.34%, indicating that black rice ATC had a certain inhibitory effect on XO. The inhibitory rate of HA (1.8 mg/mL) on XO was 40%, indicating that HA also had a certain inhibitory effect on XO. When HAA nanocomposite particles were used as inhibitors to inhibit XO activity, the inhibitory rate of HAA nanocomposite particles (2.0 mg/mL) on XO activity in vitro reached 56.42%, the inhibitory effect improved with increasing concentration. HA and black rice ATC have a synergistic effect of inhibiting XO activity effectively. Although the inhibitory effect of HAA on XO was not as strong as that of allopurinol, our results suggested that HAA is a promising XO inhibitor. In addition, many studies have found that allopurinol has strong toxicity and side effects, which can cause symptoms such as inflammatory cell infiltration of the renal interstitium and renal duct dilatation. The above results confirmed the effectiveness and applicability of HAA nanocomposite particles prepared by a simple cross-linking method in inhibiting XO, which provided a new idea for inhibiting the production of uric acid.