Comparative analysis of the inhibitory effects of aloin on tyrosinase supported by Fe₃O₄@rGO: investigation of interaction mechanisms, inhibitory activity, and conformational changes

Abstract

Tyrosinase is a key enzyme that regulates the rate of melanin synthesis, thereby modulating both food browning and skin pigmentation. It has been found that aloin is an effective inhibitor of tyrosinase activity and Fe₃O₄@rGO has remarkable drug-loading capability. In this study, enzyme inhibition kinetics and multispectral techniques were employed to investigate the enzyme inhibitory effect of Fe₃O₄@rGO-aloin nanocomposites and evaluate their anti-browning effect and safety. The binding ability of tyrosinase and aloin was further enhanced by the addition of Fe₃O₄@rGO. The IC₅₀ value of Fe₃O₄@rGO-aloin against tyrosinase was determined to be 2.26 ± 0.15 × 10⁻⁵ mol L⁻¹ with a typical anticompetitive inhibition. The findings suggest that Fe₃O₄@rGO-aloin exhibits a more potent inhibitory effect on tyrosinase compared to aloin. Furthermore, three-dimensional fluorescence, Fourier transform infrared and circular dichroism experiments demonstrated that aloin-loaded Fe₃O₄@rGO nanoparticles induce alterations in the secondary structure and conformation of tyrosinase. This indicates the potential of Fe₃O₄@rGO nanocomposites as candidates for the development of novel tyrosinase inhibitors for the treatment of hyperpigmentation disorders.

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1 Introduction

Tyrosinase (TYR) is a pivotal enzyme containing copper metal that plays a crucial role in living organisms by facilitating the production of melanin, plant pigments, and thyroid hormones. Moreover, it catalyzes the conversion of hydroxy-containing L-tyrosine to levodopa and undergoes oxidation to form dopaquinone, ultimately leading to the synthesis of melanin.^1,2 If there is an elevated concentration or excessive activity of TYR within the body, it can result in the accumulation of melanin, thereby giving rise to various dermatological conditions such as hyperpigmentation, melasma, age spots, and even skin cancer.³ Additionally, TYR plays a key role in cellular metabolism, and excessive enzymatic activity can give rise to metabolic disorders, thereby perturbing interconnected metabolic pathways and eliciting aberrant physiological responses within the organism.⁴ In vivo and cell line experiments have been used to screen for TYR inhibitors.^5,6 Therefore, the inhibition of TYR catalytic activity and the development of highly potent and low-toxicity TYR inhibitors hold immense significance in addressing pigmentation disorders and restoring normal body metabolism. Because of kojic acid's widespread use in food preservation by effectively inhibiting TYR activity, many studies have investigated the inhibitory effects of kojic acid derivatives on TYR activity.⁷ Peng et al. designed a series of gallic acid-benzylidenehydrazine hybrids and assessed their TYR inhibitory activity.⁸ He et al. synthesized 15 novel kojic acid compounds bearing 1,2,4-triazine moieties and investigated their inhibitory activities and mechanisms on TYR.⁹ Wang et al. synthesized 14 kojic acid 1,3,4-oxadiazole compounds and evaluated their TYR inhibitory activity.¹⁰ Most of the kojic acid derivatives showed better anti-tyrosinase activity than kojic acid. In addition, hydroxypyranone thiocarbazone derivatives, 4H-chromene-3-carbonitrile derivatives and thiosemicarbazide derivatives also exhibited anti-tyrosinase activity.^11,12

Meanwhile, natural compounds derived from plants, such as quercetagetin, verproside, polyphenols and so on, have also been recognized as promising sources of TYR inhibitors.^13,14 Aloin, derived from the aloe plant, has shown remarkable protective effects as an anti-tumor, anti-inflammatory,¹⁵ cytoprotective, antioxidant, antibacterial,¹⁶ anticancer,¹⁷ antidiabetic, and osteogenic agent.¹⁸ Previous work of our group confirmed that aloin has inhibitory effects on TYR.¹⁹

Due to their ability to interact at the molecular level, nanoscale particles have been used to improve the enzyme inhibitory activity of organic extracts.²⁰ Nevertheless, drug delivery systems often demonstrate a failure in the adhesion phase, resulting in the release of a high concentration of the therapeutic agent. This situation may lead to various health complications, including diminished effectiveness or liver damage. To solve these concerns, alternative methods for drug administration have been established. One notable pathway is the application of drugs on nanoporous carrier materials, such as activated carbon, graphene, and magnetic nanomaterials (Fe₃O₄). The drug encapsulation and transportation of graphene nanocomposites have been studied, as hydrophobic medications can be encapsulated onto graphene sheets through π–π stacking. Angelopoulou et al. created graphene oxide (GO)/poly(lactide)–poly(ethylene glycol) (PLA–PEG) nanocomposites containing paclitaxel, showing effective high loading capability, regulated release, and cytotoxic effects on A549 cancer cells.²¹ Zhang et al. developed a PEG-modified nano-graphene oxide to transport paclitaxel and indocyanine green, demonstrating outstanding outcomes in fluorescence tagging and treatment efficacy.²² Nanocarriers engineered to specifically bind natural drugs are employed to surmount the obstacles associated with the in vivo delivery and efficacy of small molecule drugs.²³ The US Food and Drug Administration has granted a license for the utilization of Fe₃O₄ as a drug carrier, owing to its exceptional biocompatibility, biodegradability, and minimal toxicity.²⁴ In the last ten years, GO modified with Fe₃O₄ nanoparticles has received considerable interest due to its potential uses in targeted drug delivery.²⁵ The broad P-conjugated network and the existence of various structural imperfections in the functional region of GO promote its modification with Fe₃O₄.²⁶ Owing to the ultra-fine size, biocompatibility, and magnetic behavior of Fe₃O₄ nanoparticles, these modified GO nanostructures are anticipated to effectively direct drug nanocarriers into specific cellular tissues via magnetic fields, thereby enhancing drug delivery efficiency.²⁷ In particular, when a focused magnetic field is utilized to aim at the cell site, the magnetic GO nanostructure can be guided to the cell via injection, which consequently facilitates the efficient accumulation of the drug at the targeted area.²⁸ Additionally, employing magnetic GO nanostructures can significantly improve drug availability, protect them from degradation prior to reaching targeted locations, and increase their concentration in specific cells by entering the bloodstream.²⁹ It has been proved that Fe₃O₄@rGO has good biological compatibility and safety in in vivo models.^30,31 Hence, this study presents the development of a composite drug delivery system based on graphene for magnetic targeting and drug delivery. Enzyme inhibition kinetics and multispectral techniques were employed to investigate the enzyme inhibitory effects of aloin-Fe₃O₄@rGO nanocomposites and evaluate their anti-browning effects and safety. The aim is to offer valuable insights for a deeper understanding of the loading dynamics of drug molecules and elucidate their binding mechanisms at the molecular level.

2 Materials and methods

2.1 Materials

TYR (1100 U mg⁻¹) and aloin (purity ≥ 95%) were purchased from Shanghai Yuanye Biotechnology Co. Ltd. L-Dopa (purity ≥ 99%) was obtained from McLean Biochemical Technology Co., Ltd. Stock solutions of TYR (8.32 × 10⁻⁵ mol L⁻¹), aloin (2.30 × 10⁻³ mol L⁻¹) and L-dopa (6.00 × 10⁻³ mol L⁻¹) were prepared using 0.05 mol L⁻¹ sodium phosphate buffer saline (PBS, pH 6.8). 0.86 mg mL⁻¹ Fe₃O₄@rGO suspension was prepared by dispersing 2.58 mg of Fe₃O₄@rGO black powder in 3.0 mL of double-distilled water. FeCl₃, FeCl₂, polyvinylpyrrolidone K30 (PVP), graphite powder and NH₃·H₂O were all sourced from Sinophenol Chemical Reagent Co., Ltd (SCRC, Shanghai, China) and used without further purification. PBS (pH 6.8) was prepared by mixing disodium hydrogen phosphate (Shanghai McLin Biochemical Technology Co., Ltd) and sodium dihydrogen phosphate (Sinopharm Chemical Reagents Co., Ltd). The entire stock solution was stored at 5 °C, while fresh ultra-pure water was utilized throughout all experimental procedures.

2.2 Synthesis of Fe₃O₄@rGO

The synthesis of magnetic Fe₃O₄@rGO nanoparticles was achieved via the coprecipitation method.³² First, a solution was prepared by dissolving FeSO₄·7H₂O (0.2780 g), FeCl₃ (0.3244 g) and PVP (10.0 mg) in 10.0 mL of ultra-pure water. Second, the solution was stirred at 80 °C for 30 min, followed by cooling it down to room temperature before adding ammonia water (5 mL) and GO solution (10 mL, with a concentration of 5 mg mL⁻¹). The resulting mixture was then subjected to ultrasound treatment at 25 °C for another 30 min. Finally, the mixture was transferred into a high-pressure reactor lined with a polytetrafluoroethylene container of capacity 100 ml, sealed tightly, and maintained at a temperature of 180 °C for a duration of 5 h and 30 min. After the autoclave had cooled down to room temperature, the product precipitated at the bottom of the container. The black product obtained was washed three times using ultra-pure water and ethanol solutions respectively before being dried in a vacuum drying oven at a temperature of 65 °C for a period of 12 h to yield Fe₃O₄@rGO powder. The Fe₃O₄@rGO nanomaterial was characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD). All experiments were performed at room temperature unless otherwise specified.

2.3 Ultraviolet-visible (UV-vis) experiments

An Agilent Cary 8454 UV-vis spectrometer equipped with a 1.0 cm cuvette (Thermo Fisher Technology Co., Shanghai) was utilized in this study. PBS (pH 6.8) was utilized as the blank solution. Three different combining sets of TYR, Fe₃O₄@rGO and aloin were added to 2.0 mL of PBS buffer solutions, respectively, and then three mixtures, TYR-Fe₃O₄@rGO, Fe₃O₄@rGO-aloin, and TYR-Fe₃O₄@rGO-aloin, were obtained.

2.4 Activity analysis of aloin against TYR

2.4.1 Assay for the activity of TYR. The TYR activity was determined by adapting and refining established methods from the literature, in accordance with rigorous scientific standards.³³ A series of L-dopa concentrations were prepared (1.25 × 10⁻⁴ mol L⁻¹, 2.50 × 10⁻⁴ mol L⁻¹, 5.00 × 10⁻⁴ mol L⁻¹, 1.00 × 10⁻³ mol L⁻¹, 2.00 × 10⁻³ mol L⁻¹, 3.00 × 10⁻³ mol L⁻¹, 4.00 × 10⁻³ mol L⁻¹, and 6.00 × 10⁻³ mol L⁻¹) along with a fixed concentration of TYR solution (2.08 × 10⁻⁷ mol L⁻¹) in a 2.0 mL volume. The absorbance of the reactants was measured at intervals of 10 s at 475 nm. The catalytic reaction of TYR follows the Michaelis–Menten kinetics (2.1):³⁴

The experimental data were subjected to statistical analysis using GraphPad Prism 8.0 software, and nonlinear fitting of TYR was performed based on eqn (2.1) to obtain the kinetic parameters K_m and V_max associated with the enzymatic reaction.

2.4.2 Effects of aloin and Fe₃O₄@rGO-aloin on TYR activity. The activity of TYR was quantified using the methodology previously established in our research.³⁵ The reaction system was supplemented with varying concentrations of aloin and Fe₃O₄@rGO-aloin solutions, while maintaining a fixed TYR concentration (8.32 × 10⁻⁷ mol L⁻¹). Subsequently, all samples were incubated at 30 °C in a water bath. After incubation, L-dopa was added (4.00 × 10⁻³ mol L⁻¹, substrate) and incubated for 5 min, and the absorbance was measured at 475 nm. The half maximal inhibitory concentration (IC₅₀) values of aloin and Fe₃O₄@rGO-aloin were determined based on the following conversion relationship in terms of enzyme activity:³⁶

Relative activity (%) = A₁/A₀ × 100% (2.2)

where A₁ is the reaction rate of the system with aloin and Fe₃O₄@rGO-aloin and A₀ represents the reaction rate of the system without aloin and Fe₃O₄@rGO-aloin.

2.4.3 Inhibition kinetic analysis. The types of inhibition induced by polyphenols can be divided into competitive inhibition, non-competitive inhibition, anti-competitive inhibition and mixed inhibition.³⁷ According to the above experimental method for the determination of TYR activity, a series of aloin and Fe₃O₄@rGO-aloin with different concentrations were set up to determine the rate of L-dopa oxidation catalyzed by TYR, and the double reciprocal Lineweaver–Burk plot was drawn. The inhibition types of aloin and Fe₃O₄@rGO-aloin on TYR were determined, and their inhibition constants K_i were calculated. According to the Lineweaver–Burk equation, the inhibition mechanisms are analyzed as follows:³⁸

Secondary plots were obtained from

where ν is the enzymatic reaction rate in the system; V_max denotes the maximum reaction rate; K_i is the dissociation constant of aloin and Fe₃O₄@rGO-aloin binding to the free enzyme; K_m denotes the Michaelis–Menten constant; [S] stands for the concentration of the substrate; and [I] and α represent the concentrations of the inhibitors and the apparent coefficient, respectively.

2.5 Fluorescence experiment

Fluorescence measurements were performed using an F-7100 fluorescence spectrophotometer (Hitachi, Japan) equipped with a 1.0 cm quartz colorimeter and an intelligent thermostatic water bath (Model ZC-10, Ningbo Tianhang Company, China). The scanning voltage was 450 V, the scanning speed was 1200 nm min⁻¹, and the excitation and emission silts were both 2.5 nm. The fluorescence spectra of the PBS buffer were collected at 280 nm excitation and then subtracted from the original fluorescence spectra of the sample. The internal filter and dilution effects were corrected before the binding and quenching data were analyzed.³⁹

2.5.1 Fluorescence quenching experiment. In 2.0 mL of PBS buffer solution (pH 6.8), the concentration of TYR was fixed at 2.08 × 10⁻⁷ mol L⁻¹. Without and with Fe₃O₄@rGO (6.87 × 10⁻⁴ mg mL⁻¹), aloin was continuously added from 0–4.93 × 10⁻⁵ mol L⁻¹ with intervals of 4.48 × 10⁻⁶ mol L⁻¹. The solution was incubated for 5 min, and then fluorescence quenching spectra were recorded three times at different temperatures (298 K, 301 K and 304 K).

2.5.2 Three-dimensional (3D) fluorescence assay. The 3D fluorescence spectra of TYR, TYR-Fe₃O₄@rGO and TYR-Fe₃O₄@rGO-aloin (C_TYR = 7.18 × 10⁻⁷ mol L⁻¹, C_Fe3O4@rGO = 1.10 × 10⁻³ mg mL⁻¹, and C_aloin = 3.29 × 10⁻⁶ mol L⁻¹) were recorded at a scanning speed of 2400 nm min⁻¹. The excitation wavelength is 200–400 nm (5.0 nm intervals), and the emission wavelength is 200–600 nm (1 nm intervals).

2.5.3 Synchronous fluorescence experiment. Synchronous fluorescence spectroscopic (SFS) analysis is based on previously reported methods.⁴⁰ The spectra were recorded at fixed C_TYR (5.72 × 10⁻⁷ mol L⁻¹) and C_Fe3O4@rGO (9.17 × 10⁻⁴ mg mL⁻¹), and aloin was added 11 times from 0–10.18 × 10⁻⁵ mol L⁻¹ at an interval of 9.26 × 10⁻⁶ mol L⁻¹. The fluorescence spectra at Δλ (λ_em − λ_ex) values of 15 and 60 nm were measured.

2.6 Infrared experiment

FT-IR spectra were recorded on a Tensor 27 Fourier transform infrared spectrometer (Bruker, Germany) using the attenuated total reflectance (ATR) method with a resolution of 4 cm⁻¹ and 60 scans. After the background spectra were subtracted, the FT-IR spectra of TYR, TYR-Fe₃O₄@rGO, and TYR-Fe₃O₄@rGO-aloin (C_TYR = 3.83 × 10⁻⁴ mol L⁻¹, C_Fe3O4@rGO = 0.84 mg mL⁻¹ and C_aloin = 3.30 × 10⁻³ mol L⁻¹) were determined from 1800 to 1400 cm⁻¹.

2.7 Circular dichroism (CD) experiment

The CD spectra of pure TYR (C_TYR = 2.87 × 10⁻⁶ mol L⁻¹) and TYR solutions combined with Fe₃O₄@rGO (C_Fe3O4@rGO = 0.60 mg mL⁻¹) and Fe₃O₄@rGO-aloin (C_aloin = 1.15 × 10⁻⁵ mol L⁻¹) were measured in the wavelength range of 190–250 nm using a CD spectrometer (Bio-Logic MOS 450, Claix, France) under a constant nitrogen atmosphere. The final spectra of all samples were obtained by subtracting the PBS spectrum. The secondary structure content of TYR was calculated using the online program (https://dichroweb.cryst.bbk.ac.uk/html/process.shtml).⁴¹

2.8 Time-resolved fluorescence assay

The time-resolved fluorescence decays of aloin-Fe₃O₄@rGO and aloin-Fe₃O₄@rGO-TYR were measured in a model FL920 CD fluorometer (Edinburgh Instruments, UK). The fluorescence decay monitored at 350 nm was obtained from 2 mL of aloin-Fe₃O₄@rGO solution (C_aloin = 3.14 × 10⁻⁵ mol L⁻¹, C_Fe3O4@rGO = 7.31 × 10⁻⁴ mg mL⁻¹, pH 6.8) in the absence and in the presence of TYR (1.76 × 10⁻⁷ mol L⁻¹).

3. Results and discussion

3.1 Characterization of Fe₃O₄@rGO

The nanocomposites Fe₃O₄@rGO and GO were characterized using FT-IR, XRD, and SEM. The FT-IR spectrum (Fig. 1A) revealed three distinct characteristic peaks for Fe₃O₄@rGO at 562.5 cm⁻¹, 1653 cm⁻¹, and 3459 cm⁻¹, corresponding to the stretching vibrations of Fe–O, C=C, and O–H bonds. In addition, GO exhibited characteristic peaks at 1087 cm⁻¹, 1667 cm⁻¹, and 3468 cm⁻¹, related to the tensile vibrations of C–O, C=C, and O–H bonds respectively. In comparison with the FT-IR spectrum of GO, the absence of an infrared peak at 1087 cm⁻¹ in Fe₃O₄@rGO suggested the complete reduction of GO to reduced graphene oxide (rGO).⁴² For Fe₃O₄@rGO-loaded aloin, the characteristic peaks at 553 cm⁻¹, 1235 cm⁻¹, 1608 cm⁻¹, and 3426 cm⁻¹ correspond to the stretching vibrations of Fe–O, C–O, C=C, and O–H bonds, respectively. Compared with GO and Fe₃O₄@rGO, these characteristic peaks are broadened and shifted. Meanwhile, the characteristic peaks of aloin appear in the range of 1800–2500 cm⁻¹, indicating that aloin has been successfully loaded onto Fe₃O₄@rGO.^43,44 The XRD spectrum of Fe₃O₄@rGO exhibited a characteristic pure cubic spinel crystal structure (Fig. 1B). The diffraction pattern exhibited distinct peaks at approximately 30.3°, 35.8°, 43.0°, 53.6°, 57.0°, and 62.5°, which corresponded precisely to the (220), (311), (400), (422), (511), and (440) lattice diffractions of Fe₃O₄ (JCPDS card no.19-0629).⁴⁵ The folded shape of GO could be clearly observed in the SEM image shown in Fig. 1C. In the hydrothermal synthesis of Fe₃O₄@rGO (Fig. 1D), it was evident that spherical Fe₃O₄ particles were uniformly distributed on the surface of rGO, indicating the effective prevention of Fe₃O₄ nanoparticle aggregation by synthetic Fe₃O₄@rGO.⁴⁶ Moreover, as depicted in Fig. 1E, it was evident that the distribution of aloin on the surface of Fe₃O₄@rGO exhibited a remarkable uniformity.

Fig. 1 (A) FT-IR spectra of Fe₃O₄@rGO and GO; (B) XRD spectra of Fe₃O₄@rGO; and SEM images of (C) GO; (D) Fe₃O₄@rGO; and (E) Fe₃O₄@rGO-aloin.

3.2 UV-vis absorption used to study the complex formation

UV-vis absorption spectroscopy is a straightforward and efficient technique frequently employed for investigating the formation of ligand–protein complexes. As depicted in Fig. 2A, Fe₃O₄@rGO exhibited no discernible absorption peak, while the absorption peak intensity of the TYR-Fe₃O₄@rGO complex was obviously higher than that of TYR + Fe₃O₄@rGO (the combined absorption spectra of TYR and Fe₃O₄@rGO), indicating that the interaction between TYR and Fe₃O₄@rGO affects the absorption characteristics of TYR.⁴⁷ This suggests that the fixation of TYR onto the surface of Fe₃O₄@rGO may be attributed to the substantial specific surface area of the Fe₃O₄@rGO nanomaterial. The spectra of aloin with and without Fe₃O₄@rGO were compared, revealing an increase in the UV absorption peak intensity of aloin. Additionally, a slight blue shift was observed at 262 nm, 293 nm, and 351 nm (approximately 2 nm difference: from 262/293/351 nm to 260/291/349 nm) (Fig. 2B). Moreover, the absorption peak intensity of Fe₃O₄@rGO-aloin surpassed that of Fe₃O₄@rGO + aloin, implying that the incorporation of aloin onto Fe₃O₄@rGO involved more than mere adsorption. The results indicated that the formation of the Fe₃O₄@rGO-aloin complex was facilitated by the hydrogen bonding between the hydroxyl group of aloin and the surface of Fe₃O₄@rGO. According to the formula,⁴⁸ the drug-loading capacity (DL) and encapsulation efficiency (EE) of Fe₃O₄@rGO with aloin were 30.80% and 12.64%, respectively (ESI and Fig. S1†).

Fig. 2 The UV-vis spectra of TYR-Fe₃O₄@rGO (A) and Fe₃O₄@rGO-aloin (B) (C_TYR = 2.34 × 10⁻⁷ mol L⁻¹, C_aloin = 3.89 × 10⁻⁶ mol L⁻¹, and C_Fe3O4@rGO = 5.83 × 10⁻⁴ mg mL⁻¹).

3.3 Activity analysis of aloin against TYR

3.3.1 Determination of TYR activity. The horizontal axis in Fig. 3A represented the varying concentrations of the substrate L-dopa, while the vertical axis corresponded to the maximum reaction rate. The Michaelis constant (K_m) value of the catalytic substrate L-dopa for the TYR reaction was determined to be 4.70 ± 2.4 × 10⁻⁴ mol L⁻¹ using the Michaelis–Menten equation. The maximum reaction rate (V_max) was determined to be 2.59 ± 0.30 × 10⁻⁶ mol L⁻¹ s⁻¹.

Fig. 3 (A) Catalytic reaction of TYR with the substrate L-dopa; (B) Effect of aloin and Fe₃O₄@rGO-aloin on TYR activity; (C) Lineweaver–Burk diagram of aloin inhibition of TYR; and (D) Lineweaver–Burk diagram of Fe₃O₄@rGO-aloin inhibition of TYR; C_aloin = 0.0, 2.0, 4.0 and 8.0 × 10⁻⁵; C_Fe3O4@rGO = 4.27 × 10⁻⁴ mg mL⁻¹.

3.3.2 Inhibitory effects of aloin and Fe₃O₄@rGO-aloin on TYR activity. Aloin and Fe₃O₄@rGO-aloin exhibited concentration-dependent inhibitory activity against TYR. As illustrated in Fig. 3B, different concentrations of aloin and Fe₃O₄@rGO-aloin were added to a 2 mL reaction system containing L-dopa as the substrate to investigate their impact on TYR activity. The results demonstrated a negative correlation between the relative activity of TYR and the concentration of aloin and Fe₃O₄@rGO-aloin, suggesting that both aloin and Fe₃O₄@rGO-aloin exerted inhibitory effects on TYR activity. The IC₅₀ values of aloin and Fe₃O₄@rGO-aloin for TYR can be determined from Fig. 3B as 4.04 ± 0.53 × 10⁻⁵ mol L⁻¹ and 2.26 ± 0.15 × 10⁻⁵ mol L⁻¹, respectively. The IC₅₀ value of Fe₃O₄@rGO-aloin for TYR is superior to those listed in Table S1,† suggesting that Fe₃O₄@rGO-aloin has the potential to be used as a TYR inhibitor.

3.3.3 Kinetic type of inhibition. By analyzing the Lineweaver–Burk plot, we could discern the nature of inhibition in a more intuitive and scientifically rigorous manner. When the intersection of all lines coincides with the X-axis, it indicates non-competitive inhibition. Conversely, when the intersection of all lines aligns with the Y-axis, it signifies competitive inhibition. In cases where the intersection of all lines falls within the second and third quadrants, mixed suppression is observed. Lastly, if all lines are parallel without any intersections, this corresponds to anti-competitive suppression.⁴⁹

The inhibition types of aloin and Fe₃O₄@rGO-aloin on TYR were analyzed using the Lineweaver–Burk double reciprocal diagram (Fig. 3C and D). The Lineweaver–Burk plot revealed that the lines intersected in the second quadrant with varying slopes, and as the aloin concentration increased, the plots exhibited steeper slopes, indicating a decreasing trend in maximum velocity (V_max). The results suggested a mixed inhibitory effect of aloin on TYR activity. Furthermore, the Lineweaver–Burk plot of TYR for Fe₃O₄@rGO-aloin exhibited parallelism, indicating typical anti-competitive inhibition. Compared with competitive and non-competitive inhibition, an inhibitor with anti-competitive inhibition does not directly bind to the enzyme but binds to the enzyme–substrate complex to form an enzyme–substrate–inhibitor complex (the complex cannot produce oxidative products), thereby affecting the progress of enzyme catalysis.⁵⁰ Since aloin is loaded on Fe₃O₄@rGO, it changes the inhibition of TYR by the combined effect of Fe₃O₄@rGO-aloin. The values of K_i and K_is (equilibrium constant for the binding of aloin to TYR–substrate complex, K_is = αK_i), which are crucial kinetic parameters for inhibition, can be determined by analyzing the slope (Fig. S2A†) and Y-intercept (Fig. S2B and S2C†) of the aloin/Fe₃O₄@rGO-aloin concentration. The K_i and K_is values for aloin inhibition of TYR were determined to be 3.02 × 10⁻⁵ mol L⁻¹ and 5.54 × 10⁻⁵ mol L⁻¹, respectively. However, as depicted in Fig. 3D, both the K_m value and V_max exhibited a decrease with increasing Fe₃O₄@rGO-aloin concentration. This indicates that the presence of Fe₃O₄@rGO changes the inhibition mode of aloin on TYR from the original mixed-type to uncompetitive inhibition. This suggests that Fe₃O₄@rGO-aloin cannot directly bind to free TYR, but must first bind to the TYR–substrate complex. Meanwhile, it indicates that Fe₃O₄@rGO-aloin can bind at a non-active site of TYR. Such binding may alter the enzyme's conformation, thereby further affecting its catalytic activity.⁵¹ The inhibition constants, K_{i aloin} and K_{i Fe3O4@rGO-aloin}, were determined to be 3.02 × 10⁻⁵ mol L⁻¹ and 1.52 × 10⁻⁵ mol L⁻¹, respectively. It is noteworthy that K_{i aloin} > K_{i Fe3O4@rGO-aloin}, indicating a stronger inhibitory effect of Fe₃O₄@rGO-aloin compared to aloin. The anti-browning properties of aloin and Fe₃O₄@rGO-aloin in freshly cut apples were assessed. As depicted in Fig. S3,† the control group had almost no anti-browning effect on the apple slices, while the Fe₃O₄@rGO-aloin group exhibited a more significant anti-browning effect than the aloin group. Moreover, the photographs also indicated that Fe₃O₄@rGO-aloin could prevent the apple slices from noticeable browning when stored at room temperature for up to 40 hours. This finding substantiates the preeminence of Fe₃O₄@rGO nanocomposites in the advancement of prospective whitening therapeutics or targeted drug delivery for skin lightening.⁵²

3.4 Study of fluorescence quenching

Fluorescence spectroscopy analyzes the excitation reaction, energy transfer, and collisional quenching to study the interaction between proteins and ligands. In order to investigate the interaction between TYR and aloin in the presence or absence of Fe₃O₄@rGO, fluorescence quenching experiments were employed as an effective method. The fluorescence intensity of TYR exhibited a prominent peak at approximately 348 nm (with an excitation wavelength of 280 nm), whereas both aloin and Fe₃O₄@rGO-aloin exhibited negligible fluorescence peaks (Fig. 4). With the increase of aloin/Fe₃O₄@rGO-aloin in the TYR solution, the fluorescence signal of TYR diminishes gradually, while both aloin and Fe₃O₄@rGO-aloin induce a slight blue shift in the emission peaks of TYR. The observed blue shift indicates that aloin/Fe₃O₄@rGO-aloin induces specific structural modifications in TYR, resulting in the encapsulation of its hydrophobic residues within a hydrophobic environment rather than their exposure.⁵³ Meanwhile, the fluorescence intensity of TYR gradually diminished, indicating that both aloin and Fe₃O₄@rGO-aloin interacted with TYR to quench its fluorescence. In order to further investigate the intrinsic fluorescence quenching mechanism of TYR by aloin/Fe₃O₄@rGO-aloin, the well-established Stern–Volmer and double log equations (eqn (3.1) and (3.2)) were employed. The corresponding experimental data are summarized in Table 1.⁵⁴

F₀/F = 1 + K_sv[Q] = 1 + K_qτ₀ (3.1)

Here, F₀ and F denote the fluorescence intensity of TYR prior to and subsequent to the addition of aloin/Fe₃O₄@rGO-aloin, respectively. The Stern–Volmer quenching constant, K_sv, is denoted as a measure of quenching efficiency, while the biomolecule quenching rate constant, K_q (K_q = K_sv/τ₀), represents the rate at which the fluorophore is deactivated by the biomolecule. Herein, τ₀ refers to the average lifetime of the fluorophore in the absence of a quencher and is equal to 10⁻⁸ s. Additionally, [Q] denotes the concentration of aloin/Fe₃O₄@rGO-aloin. The binding constant, K_b, and the number of binding sites, n, are essential parameters in characterizing the fluorescence quenching process. This mechanism can be categorized into dynamic quenching and static quenching. Dynamic quenching arises from collisions between the fluorophore and the quencher molecules, while static quenching predominantly results from the formation of non-fluorescent ground-state complexes between the fluorophore and the quencher. Dynamic quenching and static quenching can be distinguished by different dependences on the temperature of quenching constants or viscosity. K_sv is considered to be a method for the fluorescence quenching efficiency of quenchers. When K_sv increases with temperature, it exhibits a dynamic quenching mechanism, wherein higher temperatures lead to accelerated diffusion rates and collision-induced quenching.⁵⁵ In contrast, as the temperature increases, K_sv exhibits static quenching behavior, leading to the formation of a non-fluorescent ground-state complex through binding between the quenching molecule and the protein. According to the data obtained in Table 1, the K_sv values between aloin/Fe₃O₄@rGO-aloin and TYR decreased with increasing temperature (from 3.60 ± 0.10 × 10⁴ L mol⁻¹ to 2.91 ± 0.05 × 10⁴ L mol⁻¹ and 4.27 ± 0.14 × 10⁴ L mol⁻¹ to 3.44 ± 0.01 × 10⁴ L mol⁻¹, respectively), which means that the quenching mechanism of the two systems is static quenching, leading to the formation of ground-state complexes. In the warmed state, the static quenching increases the molecular thermal motion of the small molecules and proteins forming the ground state complex, which can effectively inhibit the activity of TYR.⁵⁶ Additionally, it is noteworthy that the K_sv value of TYR-Fe₃O₄@rGO-aloin surpasses that of TYR-aloin at identical temperatures, thereby indicating a superior fluorescence quenching effect of Fe₃O₄@rGO-aloin on TYR compared to single aloin. The K_b values of aloin and Fe₃O₄@rGO-aloin were approximately 10⁴ L mol⁻¹, indicating a moderate binding affinity for TYR in both aloin and Fe₃O₄@rGO-aloin. Additionally, the n values exhibited proximity to 1, indicating the presence of a solitary binding site on TYR.⁵⁷ In addition, the K_b values increased with temperature and were higher for Fe₃O₄@rGO-aloin compared to aloin at the same temperature, indicating a stronger binding affinity between Fe₃O₄@rGO-aloin and TYR. Furthermore, the stability of the TYR-Fe₃O₄@rGO-aloin complex also increased with temperature. The fixation of aloin onto the surface of the Fe₃O₄@rGO nanomaterial potentially enhances the likelihood of contact between TYR and aloin, leading to an elevation in the binding constant.⁵⁸

Fig. 4 (A) Fluorescence quenching spectra of the interaction between aloin and TYR; (B) fluorescence quenching spectra of aloin and TYR in the presence of Fe₃O₄@rGO. C_TYR = 2.08 × 10⁻⁷ mol L⁻¹, C_Fe3O4@rGO = 6.87 × 10⁻⁴ mg mL⁻¹, and C_aloin = 0–49.28 × 10⁻⁶ mol L⁻¹ (with an interval of 4.48 × 10⁻⁶ mol L⁻¹, 12 times). The inset figures in Fig. 4 is the quenching effect graphs of the Stern–Volmer equation.

Table 1 The binding constants and thermodynamic parameters of TYR-aloin and TYR-(Fe₃O₄@rGO-aloin)

T (K)	Ksv (× 10^4 L mol^−1)	R^2	n	Kb (× 10^4 L mol^−1)	ΔG° (kJ mol^−1)	ΔH° (kJ mol^−1)	ΔS° (J mol^−1 K^−1)
TYR-aloin
298	3.60 ± 0.10	0.9933	0.98	2.35	−25.01	182.57	696.56
301	3.21 ± 0.06	0.9969	1.06	5.35	−27.10
304	2.91 ± 0.05	0.9971	1.12	10.10	−29.19
TYR-(Fe3O4@rGO-aloin)
298	4.27 ± 0.14	0.9905	1.14	7.72	−27.88	42.20	235.15
301	3.67 ± 0.12	0.9907	1.04	9.09	−28.59
304	3.44 ± 0.01	0.9926	1.12	10.80	−29.29

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In general, small molecules bound to biological macromolecules are subject to four interaction forces: hydrophobic interactions, electrostatic forces, hydrogen bonding, and van der Waals forces.⁵⁹ The type of force can be determined by analyzing the positive and negative enthalpy change (ΔH°) and entropy change (ΔS°) in thermodynamic parameters. Therefore, the primary driving force behind the synergistic effect of aloin or Fe₃O₄@rGO-aloin in combination with TYR can be characterized by the thermodynamic parameters derived from the well-established van't Hoff equations:

ln K_b = −ΔH°/RT + ΔS°/R (3.3)

ΔG° = ΔH° − TΔS° (3.4)

where ΔH°, ΔS°, ΔG°, R and T stand for enthalpy change, entropy change, free energy change, gas constant (8.314 J mol⁻¹ K⁻¹) and temperature, respectively. The negative values of ΔG° indicated that aloin and Fe₃O₄@rGO-aloin interactions with TYR were both spontaneous processes, and the positive values of ΔS° indicated that the aloin-TYR/Fe₃O₄@rGO-aloin-TYR systems become more disordered. Moreover, the positive ΔH° and ΔS° values indicated that hydrophobic action played a major role in the formation and stabilization of the aloin-TYR/Fe₃O₄@rGO-aloin-TYR complex,⁶⁰ and hydrogen bonds may also be involved in the binding due to its polyhydroxyl structure.

3.5 Excitation–emission matrix analysis

The 3D fluorescence spectrum enables the comprehensive visualization of chromophore fluorescence information, thereby enhancing the scientific investigation of protein conformational changes. The excitation–emission matrix fluorescence spectra of TYR exhibited variations upon interaction with Fe₃O₄@rGO-aloin, indicating the presence of an interaction between Fe₃O₄@rGO-aloin and TYR (Fig. 5). Peaks 1 and 2 were Rayleigh scattering peaks (λ_em = λ_ex) and second-order scattering peaks (λ_em = 2λ_ex), respectively. Peak a mainly represented the fluorescence properties of the polypeptide backbone. Peak b essentially stood for the spectral behavior of tryptophan and tyrosine residues.⁶¹

Fig. 5 Three-dimensional fluorescence spectra. (A) TYR; (B) TYR-Fe₃O₄@rGO; and (C) TYR-(Fe₃O₄@rGO-aloin). C_TYR = 7.18 × 10⁻⁷ mol L⁻¹, C_aloin = 3.29 × 10⁻⁶ mol L⁻¹ and C_Fe3O4@rGO = 1.10 × 10⁻³ mg mL⁻¹.

After the introduction of Fe₃O₄@rGO into TYR, slight shifts in the positions of peak a and peak b were observed (Table 2). The fluorescence intensities of peak a and peak b exhibited minimal changes, with decreases of 13.74% and 7.48%, respectively. This suggests that the conformational change of TYR was minimally affected by Fe₃O₄@rGO, indicating the excellent biocompatibility and low toxicity of Fe₃O₄@rGO as a potential drug carrier.⁶² However, upon the addition of Fe₃O₄@rGO-aloin to the TYR solution, peak a exhibited a decrease in intensity from 1093 to 688.3 along with a shift in its position. This observation signifies perturbation of the peptide backbone and alteration of the microenvironment resulting from binding.⁶³ Additionally, when combined with Fe₃O₄@rGO-aloin, the intensity of peak b decreases from 660.5 to 489.8, and the Stokes shift varies slightly, suggesting that Fe₃O₄@rGO-aloin influences the secondary structure of TYR. Therefore, it can be postulated that the interaction with Fe₃O₄@rGO-aloin perturbed the polypeptide backbone, resulting in a more relaxed conformation.⁶⁴

Table 2 Analysis of three-dimensional spectral data of TYR, TYR-Fe₃O₄@rGO and TYR-(Fe₃O₄@rGO-aloin)

Compounds	Peak a	Δλ	Intensity F	Peak b	Δλ	Intensity F
TYR	225/354	129	1093	280/352	72	660.5
TYR-Fe3O4@rGO	225/351	126	942.8	280/353	73	611.1
TYR-(Fe3O4@rGO-aloin)	225/350	125	688.3	280/351	71	489.8

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3.6 Conformational alterations of TYR in its binding with aloin in the presence and absence of Fe₃O₄@rGO

SFS was employed to characterize the impacts of Fe₃O₄@rGO-aloin on the hydrophobicity and polarity of tyrosine (Tyr) and tryptophan (Trp) residues within TYR. The fluorescence quenching of TYR by Fe₃O₄@rGO-aloin revealed a significant impact of Fe₃O₄@rGO-aloin on the microenvironment surrounding TYR. The SFS at Δλ = 15 nm and 60 nm were utilized to investigate the polarity changes in the microenvironments of Tyr and Trp residues, respectively (Fig. 6A and B).⁶⁵ The fluorescence intensities of Tyr and Trp gradually diminished with the continuous addition of Fe₃O₄@rGO-aloin, indicating their interaction with Fe₃O₄@rGO-aloin and the subsequent quenching of internal fluorescence. Simultaneously, the addition of Fe₃O₄@rGO-aloin did not lead to the shift of the fluorescence peak of the Tyr residue; however, it resulted in a blue shift of the fluorescence peak of the Trp residue from 285 nm to 282 nm (∼3 nm). This observation suggested that Fe₃O₄@rGO-aloin did not have a noticeable effect on the surroundings of Tyr residues, but it could potentially lead to changes in the environment surrounding Trp residues. Therefore, these findings qualitatively emphasize the impact of the Fe₃O₄@rGO-aloin-TYR interaction on the conformation and microenvironment of TYR. Moreover, the values of the ratio of synchronous fluorescence quenching (RSFQ), calculated by virtue of the equation RSFQ = 1 − F/F₀, were used to compare the contribution of Tyr and Trp residues in fluorescence quenching (Fig. 6C). The results revealed that the RSFQ values at Δλ = 15 nm and 60 nm nearly overlap for Fe₃O₄@rGO-aloin, signifying that the Tyr and Trp residues contribute approximately equal amounts to TYR fluorescence quenching.⁶⁶

Fig. 6 (A) (Δλ = 15 nm) and (B) (Δλ = 60 nm) show synchronous fluorescence spectra of TYR and aloin in the presence of Fe₃O₄@rGO. C_TYR = 5.72 × 10⁻⁷ mol L⁻¹, C_Fe3O4@rGO = 9.17 × 10⁻⁴ mg mL⁻¹, and C_aloin = 0–10.18 × 10⁻⁵ mol L⁻¹ (with an interval of 9.26 × 10⁻⁶ mol L⁻¹, 11 times) corresponding to curves 1 → 12, respectively. (C) Comparative evaluation of the impact of Fe₃O₄@rGO-aloin on the synchronous fluorescence quenching ratios (RSFQ) of TYR.

FT-IR was used to study the secondary structure of TYR. There were two typical amide bands in the infrared spectrum of proteins, mainly amide I band (1700–1600 cm⁻¹) and amide II band (1600–1500 cm⁻¹). The former mainly reflected the C=O stretching property, while the latter mainly reflected the C–N stretching and N–H bending. This was a prominent feature of protein groups in Fourier transform infrared spectroscopy, which was of great significance for determining the composition and conformational change of the protein secondary structure. Since the amide I band (1600–1700 cm⁻¹) played a major role in the secondary structural changes of proteins, the amide I band was more frequently used to evaluate the secondary structural changes of proteins. With the addition of Fe₃O₄@rGO, the characteristic absorption peaks of the amide I and amide II bands of TYR did not shift significantly (Fig. 7A). However, with the addition of Fe₃O₄@rGO-aloin, the characteristic absorption peaks of amide I and amide II bands of TYR shifted to different degrees (the amide I band shifted from 1645 cm⁻¹ to 1632 cm⁻¹ and the amide II band moved from 1533 cm⁻¹ to 1537 cm⁻¹). The results suggested that Fe₃O₄@rGO had little effect on the structure of TYR, while Fe₃O₄@rGO-aloin had a certain extent effect on the changes in the functional groups (C=O and C–N) of the structure of TYR, which changed the conformation of TYR and affected the catalytic activity of TYR.

Fig. 7 (A) Infrared spectra of free TYR, TYR-Fe₃O₄@rGO and TYR-Fe₃O₄@rGO-aloin (C_TYR = 3.83 × 10⁻⁴ mol L⁻¹, C_Fe3O4@rGO = 0.84 mg mL⁻¹, and C_aloin = 3.30 × 10⁻³ mol L⁻¹). (B, C and D) show the curve-fitted amide I region of free TYR, TYR-Fe₃O₄@rGO, and TYR-(Fe₃O₄@rGO-aloin), respectively.

To determine the change in the secondary structure, the curve-fittings were used to analyze the amide I band. The amide I band can be divided into five band ranges and the spectral ranges of 1660–1650 cm⁻¹, 1680–1660 cm⁻¹, 1692–1680 cm⁻¹, 1648–1638 cm⁻¹ and 1637–1610 cm⁻¹ were due to α-helix, β-turn, β-antiparallel, random coil and β-sheet structure, respectively. As shown in Fig. 7B, the free TYR contained 26.97% α-helix, 23.59% β-turn, 9.59% β-antiparallel, 22.91% random coil and 16.48% β-sheet. The conformation of TYR did not change much with the addition of Fe₃O₄@rGO (Fig. 7C). With the addition of [Fe₃O₄@rGO-aloin] : [TYR] (from 0 : 1 to 8 : 1), a decreasing tendency of the contents of α-helix, random coil, and β-turn tended to decrease (from 26.97% to 14.55%, from 22.91% to 16.46% and from 23.59% to 14.41%, respectively), while the contents of β-sheet and β-antiparallel tended to increase (from 16.48% to 43.35% and from 9.59% to 11.23%) (Fig. 7D). These results imply that the interaction between Fe₃O₄@rGO-aloin and TYR modifies the polypeptide chain and eventually causes a transformation in the conformation of TYR. Consequently, it is believed that Fe₃O₄@rGO-aloin exhibits anti-tyrosinase activity by interacting with TYR and disrupting the structure.

3.7 CD spectroscopy

To investigate the effect of aloin on the secondary structure of TYR, the CD spectra (190–240 nm) of TYR were measured in the presence and absence of Fe₃O₄@rGO. As shown in Fig. 8, the CD intensity of the characteristic peaks of TYR changed little with the addition of Fe₃O₄@rGO. However, the CD intensity of the characteristic peaks of TYR increased with the addition of Fe₃O₄@rGO-aloin. This indicates that Fe₃O₄@rGO has little effect on the secondary structure of TYR, while Fe₃O₄@rGO-aloin, due to its interaction with TYR, changes the secondary structure of TYR. As shown in Table 3, free TYR contains 22.3% α-helix, 29.7% β-turn, 22.1% random coil, and 26.9% β-sheet. After the addition of Fe₃O₄@rGO, the conformation of TYR does not change significantly. With the addition of Fe₃O₄@rGO-aloin, the contents of α-helix, random coil, and β-turn show a downward trend (from 22.3% to 12.8%, from 21.1% to 17.5%, and from 29.7% to 20.2%, respectively), while the content of β-sheet shows an upward trend (from 26.9% to 49.5%). These results indicate that after binding with Fe₃O₄@rGO-aloin, the hydrogen bond network between the helical structures of TYR is disrupted, leading to a more relaxed conformation of TYR. It was reported that the content of α-helix was inversely proportional to TYR activity, the greater the conformational change induced by the inhibitor, the stronger the inhibitory ability.⁶⁷ Fe₃O₄@rGO-aloin inhibits TYR activity by altering its spatial structure and causing it to lose its normal physiological function.

Fig. 8 CD spectra of the interaction between Fe₃O₄@rGO or Fe₃O₄@rGO-aloin and TYR at 298 K (C_TYR = 2.87 × 10⁻⁶ mol L⁻¹, C_Fe3O4@rGO = 0.60 mg mL⁻¹, and C_aloin = 1.15 × 10⁻⁵ mol L⁻¹).

Table 3 The secondary structure content of TYR in the presence and absence of Fe₃O₄@rGO/Fe₃O₄@rGO-aloin

Compounds	α-Helix (%)	β-Sheet (%)	β-Turn (%)	Random coil (%)
TYR	22.3	26.9	29.7	21.1
TYR-Fe3O4@rGO	21.6	25.3	32.6	20.5
TYR-Fe3O4@rGO-aloin	12.8	49.5	20.2	17.5

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3.8 Time-resolved photoluminescence of aloin-Fe₃O₄@rGO and aloin-Fe₃O₄@rGO-TYR

Generally, the most definitive method to distinguish between static and dynamic quenching is the fluorescence lifetime. The non-variance of the lifetime (τ) values points to a static fluorescence quenching mechanism, while the substantial decrease of the τ values reveals the appearance of a dynamic quenching process. The interaction of their lifetimes at excitation/emission wavelengths of 280/350 nm was investigated to gain insight into the quenching mechanism between TYR and Fe₃O₄@rGO-aloin (Fig. 9). Table 4 showed that the fluorescence lifetimes of aloin-Fe₃O₄@rGO (τ₁ = 0.62 ± 0.01 ns and τ₂ = 5.37 ± 0.52 ns, with relative percentages of 89.36% and 12.11%, respectively χ² = 1.3002) and aloin-Fe₃O₄@rGO-TYR (τ₁ = 0.71 ± 0.01 ns and τ₂ = 5.44 ± 0.46 ns, with relative percentages of 86.45% and 14.89%, χ² = 1.1874) were the same within the experimental error. This confirmed that the fluorescence quenching mechanism was a static process.⁶⁸

Fig. 9 Time-resolved fluorescence decays for Fe₃O₄@rGO-aloin without and in the presence of TYR. C_TYR = 1.76 × 10⁻⁷ mol L⁻¹, C_aloin = 3.14 × 10⁻⁵ mol L⁻¹ and C_Fe3O4@rGO = 7.31 × 10⁻⁴ mg mL⁻¹.

Table 4 Time-resolved fluorescence parameters of aloin-Fe₃O₄@rGO and aloin-Fe₃O₄@rGO-TYR

Compounds	τ1 (ns)	A1%	τ2 (ns)	A2%	τavg (ns)	χ^2
A: relative amplitude. χ^2: the fitting correlation coefficient.
Aloin-Fe3O4@rGO	0.62 ± 0.01	89.36	5.37 ± 0.52	12.11	3.19	1.3002
Aloin-Fe3O4@rGO-TYR	0.71 ± 0.01	86.45	5.44 ± 0.46	14.89	3.40	1.1874

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4 Conclusions

Enzyme inhibition kinetics analysis and multispectral approaches were employed to investigate the interaction between Fe₃O₄@rGO-loaded aloin and TYR. Kinetic studies revealed that the IC₅₀ value of aloin and Fe₃O₄@rGO-aloin on TYR were (4.04 ± 0.53) × 10⁻⁵ mol L⁻¹ and (2.26 ± 0.15) × 10⁻⁵ mol L⁻¹, respectively. The experimental results clearly demonstrated that the inhibitory effect of Fe₃O₄@rGO-aloin on TYR was more powerful than that of single aloin. From the Lineweaver–Burk diagram, it was evident that aloin and Fe₃O₄@rGO-aloin exhibit mixed inhibition and anticompetitive inhibition on TYR, respectively. The inhibition of TYR and the interaction mechanism of Fe₃O₄@rGO-loaded aloin were determined to be a static quenching process, primarily reliant on hydrophobic interaction. Aloin exhibited a moderate binding capacity to TYR, with a binding constant of approximately 10⁴ L mol⁻¹. Furthermore, it was found that the combined ability of TYR and aloin was further enhanced by the addition of Fe₃O₄@rGO. The impact of Fe₃O₄@rGO-aloin on the microenvironment of TYR was demonstrated through synchronous fluorescence experiments. The impact of Fe₃O₄@rGO-aloin on the secondary structure of TYR was observed to be more pronounced compared to that of Fe₃O₄@rGO, as evidenced by 3D fluorescence, FT-IR and CD experiments. The findings of this study yield valuable insights for the design of functional magnetic reduced graphene oxide. Specifically, they offer a deeper understanding of the molecular-level interaction mechanism between Fe₃O₄@rGO-supported aloin and TYR. Such information is crucial for advancing research in this field and developing novel materials with enhanced properties.

Author contributions

Zhu Wang: data curation, investigation, software, formal analysis, and writing – original draft. Lu Chen: software, validation, formal analysis, writing – reviewing and editing, and visualization. Jing Yuan: investigation, data curation, validation, and formal analysis. Qiulan Zhang: conceptualization, methodology, supervision, funding acquisition, and writing – reviewing and editing. Yongnian Ni: visualization, writing – reviewing and editing, resources, and supervision.

Conflicts of interest

There are no conflicts of interest to declare.

Data availability

The authors confirm that the data supporting the findings of this study are available within the article. Additional data that support the findings of this study are available from the corresponding author, QLZ, upon reasonable request.

Acknowledgements

The authors gratefully acknowledge the financial support from the National Natural Science Foundation of China (No. 21665017) and the Science and Technology Innovation Platform Project of Jiangxi Province (20192BCD40001).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5pm00067j

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