1. Introduction
Hispidin was first extracted from the sarcocarp body of the
Inonotus hispidus fungus in 1889, and its structure was subsequently elucidated to be 6-(3,4-dihydroxyphenyl)-4-hydroxy-2-pyrone in 1961 [
1,
2]. As a secondary metabolite, hispidin is found in numerous fungus families, including
Hymenochaetaceae [
3],
Hymenogastraceae [
4],
Phaeolaceae [
5],
Omphalotaceae [
6], and
Cortinariaceae [
7]. Additionally, several plants, such as
Equisetum arvense [
8],
Pistacia atlantica [
9],
Leishmania amazonensis [
10], and
Goniothalamus umbrosus [
11], also contain hispidin and its derivatives [
12]. Hispidin is attracting the attention of researchers for its potential in preventing and treating cancer [
13], cardiovascular disease [
14], neurodegenerative illnesses [
15], and viral infections [
16]. Hispidin has been shown to have anti-inflammatory, anti-oxidant, antiallergic, antiangiogenic, and hypoglycemic properties in earlier studies. Recently, the antidiabetic properties of hispidin have been observed to inhibit ferroptosis, thereby safeguarding pancreatic beta cells (PBCs) from damage induced by excessive glucose [
17]. Although there are many potential health benefits associated with hispidin, further research is needed to determine whether or not it is safe for human usage.
The extent to which exogenous substances bind to blood albumin dictates their distribution and metabolism [
18,
19,
20]. Through their reversible interaction with plasma proteins, such as human serum albumin (HSA), certain drugs, food additives, and other bioactive small molecules can alter their distribution, free concentration, and metabolism [
21,
22,
23]. In addition, reducing aggregate formation and generally lengthening a drug’s half-life, HSA binding has exceptional effects on drug transport, release, and solubility [
24]. HSA is one kind of soluble protein that is widely available in plasma; it contributes to 80% of osmotic blood pressure [
25], and binds and moves naturally occurring hydrophobic ligands. HSA is a protein with 585 amino acid residues and a molecular weight of 66,500 Da [
26]. It makes up around 4.5% of the mass of blood in humans and aids in regulating the pH and osmotic pressure of blood [
27]. In a physiologically healthy state, its blood concentration falls between 0.5 and 0.75 mM (35 and 50 mg/mL) [
28]. Three homologous domains, I (residues 1–195), II (residues 196–383), and III (residues 384–584), are present in HSA. Each domain is divided into two subdomains, A and B, which share sequence amino acids. Sudlow’s sites I and II are the main places where HSA combines, which reside in particular gaps in subdomains IIA and IIIA, respectively, and a few subordination places (site III or digitoxin sites), and are largely responsible for HSA’s ability to combine some aromatic and heterocyclic ligands [
29]. Site I is a pocket that arises within subdomain IIA. It is also known as the warfarin-azapropazone site [
30]. The lone tryptophan (Trp214) in the protein is found there. Subdomain IIIA contains site II, which has an affinity for ibuprofen. Hydrophobic amino acid residues are present in the interior pocket, whereas two crucial residues of amino acids (Arg410 and Tyr411) are present in the exterior pocket. Moreover, hemin is a site probe for domain I [
31]. Additionally, other research has discovered that fatty acids can be used as probes to locate the binding site in HSA subdomain IIA [
32].
In this work, the interaction between hispidin and HSA was investigated using various methodologies, including fluorescence quenching, synchronous fluorescence, three-dimensional fluorescence, UV/vis spectroscopy, hydrophobic probe assays, and site competition experiments. The quenching mode, the apparent binding constant, the quantity of binding sites, and the thermodynamic constants of the mutual effect of hispidin with HSA were determined. Finally, to validate the binding site and binding modalities of hispidin with HSA, a molecular docking simulation was performed. This work provides fundamental data to elucidate the binding mechanism of hispidin with HSA, which can aid in comprehending the pharmacological or toxicological effects of hispidin.
3. Discussion
Vitamins, hormones, steroids, fatty acids, and other endogenous and exogenous compounds (drugs, toxins, phytochemicals) are among the diverse substances that HSA is essential in binding and conveying. Certain small compounds included in food have the ability to bind to HSA and change its conformation or spatial configuration. On the contrary, HSA may have an influence on small molecule metabolism and their effective concentration in the body. Consequently, research on the interaction between hispidin and HSA is crucial for the fields of chemistry, medicine, biology, and food nutrition and health.
Due to its great sensitivity, simplicity in use, and speed, fluorescence spectroscopy is a commonly used method for determining non-covalent interactions between proteins and small molecules [
41]. The static and dynamic quenching mechanisms are distinguished according to temperature dependence [
42]. In this study, hispidin exhibited the ability to quench the fluorescence of HSA, and the static quenching characteristic was quantified using the Ksv value, which consistently decreased with an increase in temperature. This effect aligns with previous observations during the interaction between 4-(1h-indor-3-yl)-2-(ptolyl) quinazoline-3-oxide and human serum albumin [
43]. Another method to investigate the quenching process is to utilize the maximal biomolecular scattering collision quenching constant (Kq). A drop in fluorescence intensity induced by static quenching occurs when the Kq value exceeds 2 × 10
10 L/(mol∙s). Otherwise, the dynamic quenching of protein binding occurs. The information in
Table 1 demonstrates that the Kqs ranged from 7.37 to 4.67 × 10
12 L/(mol·s) at various temperatures, all of which were higher than 2 × 10
10 L/(mol·s), also suggesting that the static quenching process is responsible for the interaction between hispidin and HSA. Tryptophan (Trp) and tyrosine (Tyr) residues are the primary sources of intrinsic fluorescence in proteins. These residues can be quenched following interactions with ligands, which alters the protein’s intrinsic fluorescence intensity [
44]. The characteristic information of Tyr or Trp is determined through synchronous fluorescence when the wavelength intervals (Δλ) are stabilized at 15 nm or 60 nm, respectively [
45]. In this study, the synchronous fluorescence spectra showed that adding hispidin caused a decrease in the fluorescence intensity of HSA (
Figure 3). The results suggest that the microenvironments of Tyr and Trp undergo changes due to chemical interactions, leading to the hispidin-induced fluorescence quenching of HSA.
Hispidin is a phenolic chemical that is a member of the C=C-bond-type bicyclic aromatic compounds [
46]. Under non-oxidizing conditions, phenolic compounds and plasma proteins form reversible complexes are mediated by hydrogen bonding, electrostatic interactions, hydrophobic effects, and van der Waals forces [
47]. Thermodynamic characteristics like ∆H and ∆S influence the way phenolic compounds interact with proteins. Hydrophobic interaction is indicated by positive values of both parameters; van der Waals forces and hydrogen bonds are indicated by negative values; and electrostatic forces are significant in aqueous solutions, as indicated by ∆S > 0 and ∆H < 0. In the results of this study, both ∆S and ∆H are positive, suggesting that the hydrophobic interaction between the aromatic ring of hispidin and the hydrophobic amino acid residues of HSA is the primary force stabilizing the hispidin–HSA interaction. In the interaction between the albumins and chemicals, the aromatic ligands are important. Hydrophobic linkages are created when the aromatic ring of a polyphenol is attracted to hydrophobic areas of other substances. If the complexes have a more hydrophobic character due to the presence of aromatic ligands, the hydrophobic interaction between the complexes and albumins is prominent. In hispidin with two aromatic rings, it makes sense to think of hydrophobic interactions as the main types of interactions. The two main non-covalent forces involved in phenol–protein interactions are often hydrogen bonding and hydrophobic interactions. This is consistent with previous findings that the binding between naringenin or genistein and HSA is strongly involved in hydrophobic interactions [
48,
49]. To verify the hydrophobic interaction between hispidin and HSA, we also employed SDS and urea as the reagents. As urea functions as a hydrogen bond receptor, it can break both hydrophobic and hydrogen bonds in proteins, causing the proteins to unfold, whereas hydrophobic connections can be entirely or partially broken by SDS, an anionic detergent [
50]. Therefore, the hydrogen bonds and hydrophobic interactions between ligands and proteins can be broken by urea and SDS, respectively. The information in
Table 3 demonstrates that urea and SDS reduced the Ka of hispidin–HSA, indicating that hydrophobic interactions are crucial for hispidin’s binding to HSA.
Due to its high affinity for the hydrophobic surface of proteins, ANS is used as a probe for hydrophobic fluorescence. It is also used as a microenvironment probe for proteins because of its exceptional capacity to exhibit peak shifts and intensity variations in response to the solvent environment in which it is found [
51]. A more thorough explanation of the hydrophobic clusters can be obtained from the fluorescence of ANS attached to proteins [
52]. In this study, the role of hydrophobic force in the hispidin–HSA interaction was proven again by employing ANS to measure the surface hydrophobicity (
S0) of HSA (
Table 5).
To further explore the binding position of the hispidin–HSA interaction, we conducted a site-competition test and molecular docking simulation. It can be seen from
Table 6 that when ibuprofen and warfarin are present, the Ka value is reduced by 32.5% and 42.7%, respectively (
Table 6). Large-volume heterocyclic anions with charges located at the center of the molecule (e.g., warfarin) typically attach to site I of the HSA, while site II is where aromatic carboxylic acids, like ibuprofen, that have prolonged conformations and negative charges at one end of the molecule interact, which can be found in the hydrophobic cavities of HSA subdomains IIA and IIIA, respectively [
53]. Thus, hispidin binds to ibuprofen and warfarin in the same location, particularly occupying site I of the warfarin-bound IIA subdomain. In domain IIA, the outcomes of molecular docking were also displayed, and the primary amino acid residues Cys245(A) and Leu250(A) were crucial to the interaction process between HSA and hispidin (
Figure 7). This also confirms that the binding between hispidin and HSA is hydrophobic. Hispidin belongs to the styrene-pyranone family of compounds, which have similar effects to flavonoids in plants [
54]. For instance, flavonol primarily binds to the IIA subdomain in HSA, and quercetin binds to a sizable hydrophobic cavity within the IIA subdomain, as indicated by a computational map of potential binding locations [
55]. Curcumin and genistein primarily bind within the hydrophobic pocket at site I of Tyrosine 214 [
56]. Gallic acid was found to be able to bind to HSA’s site I in a different investigation [
57]. Phenolic compounds typically undergo high first-pass metabolism. Further investigation is warranted in the future to explore the interaction between hispidin primary metabolites and HSA.
4. Materials and Methods
4.1. Materials
HSA was purchased from Solarbio Biotechnology Co. Ltd. (Beijing, China). Hispidin (purity ≥ 98%), digitoxin (purity ≥ 98%), hemin (purity 98%), ibuprofen (purity ≥ 98%), warfarin (purity ≥ 98%), and 8-anilino-1-naphthalenesulfonic acid (ANS, purity 96%) were obtained from Macklin Biochemical Technology Co. Ltd. (Shanghai, China). Digitoxin, hemin, ibuprofen, and warfarin were used for binding site exploration. SDS and urea were purchased from Biosharp Reagent Co. Ltd. (Hefei, China) and Aladdin Biochemical Technology Co. Ltd. (Shanghai, China), respectively. The remaining reagents used were of analytical grade.
4.2. Preparation of Reaction Solutions
The concentration of Tris-HCl buffer solution was 0.2 mol/L, which included 0.1 mol/L NaCl, and the value of pH was kept at 7.4. Prior to use, the HSA stock (0.2 mmol/L) was diluted with Tris-Cl buffer solution, and the final HSA concentration was 5 µmol/L. In order to generate 40 mmol/L solutions, hispidin was dispersed in DMSO and maintained in a brown bottle. The interaction between hispidin and HSA was investigated using various methodologies, including fluorescence quenching, synchronous fluorescence, three-dimensional fluorescence, UV/vis spectroscopy, hydrophobic probe assays, and site competition experiments. Before the experiment, the stock solution was diluted with Tris-HCl to generate a working solution (5 mmol/L). The working hispidin solutions were added to a solution of HSA to generate different hispidin concentrations (5–45 µmol/L), with the DMSO content at the end being less than 0.01%. Additionally, to confirm how SDS and urea affect the interaction between HSA and hispidin, SDS and urea, which had been dissolved in Tris-Cl, were added to the HSA–hispidin mixed solution at final concentrations of 4 mol/L and 5 mmol/L, respectively. The fluorescence intensity values of the samples were measured in quartz glass cuvettes after a 5 min reaction period at four different temperatures (298, 303, 310, and 313 K).
4.3. Fluorescence Spectroscopy Analysis
Using an F97XP fluorescence spectrophotometer (Shanghai, China), the fluorescence spectrum of the HSA solutions, either with or without hispidin, SDS, and urea, was examined. The HSA concentration in all the experiments was 5 µmol/L. The emission spectra were obtained in the wavelength range of 300–420 nm using an excitation wavelength of 280 nm and a scanning speed of 1000 nm/min. The fixed slit widths for both emission and excitation were set at 5 nm. Tris-Cl buffer solution was used as a blank before the measurement.
4.3.1. Identification of the Fluorescent Quenching Process
Two mechanisms were considered for the process of quenching fluorescence, which is the reduction in a fluorophore’s intensity: static and dynamic quenching. The mechanisms underlying fluorescence quenching were elucidated by employing the Stern–Volmer Equation (1) [
58].
In Equation (1), F0 and F represent fluorescence intensity values without and with a quencher, respectively, Ksv stands for the quenching constant of Stern–Volmer, and Kq denotes the rate constants of quenching. Without a quencher, δ0 represents a molecular fluorescence lifetime of 10
−8 s [
59], and [Q] is the quencher concentration. A plot of F0/F versus [Q] was used to obtain Ksv from the slope of linear regression. In cases when the Kq value exceeds 2 × 10
10 L/(mol·s), static quenching is the reason for the decreased fluorescence intensity; if not, there is dynamic quenching for the binding of the quencher protein.
4.3.2. Ka Measurement and Site Numbering
During static quenching, small molecules individually bind to specific sites on a macromolecule [
60]. To obtain the binding sites and Ka value between hispidin and HSA, the Lineweaver–Burk equation was created by modifying the Stern-Volmer equation (Equation (2)) [
61].
In this formula, the fluorescence intensity when the quenching agent is present is F, and when it is not, it is F0, and [Q] is the concentration of the quenching agent.
4.3.3. Determining Evident Thermodynamic Parameters
The Van ’t Hoff equation (Equation (3)) was utilized to determine the apparent enthalpy and entropy changes (∆H and ∆S) by analyzing the slope and intercept of the lgKa versus 1/T curve; however, using Equation (4), the standard free energy change G
◦ of hispidin binding to HSA was computed [
62].
R is the gas constant, which is 8.314 J/(mol K), and T stands for thermodynamic temperature, also known as the absolute temperature, T = t (°C) + 273.15. Ka is calculated from Formula (2). Generally, non-covalent interactions between small molecules and biological protein macromolecules involve four non-covalent interaction forces. The hydrophobic force acts as the primary driving force when ∆H > 0 and ∆S > 0; Van der Waals and hydrogen bonding forces predominate if ∆H < 0 and ∆S < 0; however, the contact is controlled by electrostatic force if ∆H < 0 and ∆S > 0 [
63].
4.4. Synchronous Fluorescence Analysis
The alterations in luminous amino acids during the interaction of proteins with small molecules were observed using synchronous fluorescence spectroscopy. Based on the equation Δλ = λem − λex, the spectral properties of protein tyrosine and tryptophan residues are displayed in the fluorescence spectra at Δλ = 15 nm and Δλ = 60 nm, respectively. In this paper, the fluorescence spectra of HSA interacting with hispidin were scanned using this method. The fixed slit widths for both emission and excitation were set at 5 nm.
4.5. UV/vis Spectroscopy Analysis
Different hispidin concentrations (0–45 µmol/L) were obtained by combining the HSA solution (5 µmol/L) with the hispidin working solutions and carrying out a reaction at room temperature for 5 min. The UV absorption peak at 220–460 nm was measured for the HSA mixes and hispidin (45 µmol/L, without HSA) with a 0.1 nm sampling interval. Before the measurement, the baseline of the device was adjusted using the Tris-HCl buffer.
4.6. Three-Dimensional Fluorescence Analysis
With or without an equimolar hispidin concentration, the three-dimensional fluorescence spectra of the HSA were recorded at a scanning rate of 48,000 nm/min at 298 K. A range of 200–900 nm was employed for both the emission and excitation wavelengths. Ten nanometers was the fixed slit width for both the emission and excitation.
4.7. Hydrophobic Probe Assay and Hydrophobicity Measurements
In the hydrophobic probe analytical experiments, the first system maintained a constant HSA level of 5 µmol/L, while in the second system, the concentration ratio of the HSA–hispidin mixture was 1:3. Subsequently, increasing titration of the ANS solution (5 × 10−3 mol/L) was added to the first system or second system solutions. The ANS concentration varied from 5 to 45 µmol/L. In the hydrophobicity experiments, initially, three systems of compounded solutions were prepared with concentration ratios ([hispidin]/[HSA]) of 0, 1, and 3, respectively. Subsequently, a 1 μL mixed solution was titrated into the 1 mL ANS solution (9 × 10−7 mol/L) sequentially. Following each addition, all experiments were allowed to react for five minutes in the dark. The excitation wavelength for the ANS fluorescence spectra was 370 nm, and the measurements were conducted within the wavelength range of 400 to 600 nm. Lastly, a plot was generated using the HSA concentration against the fluorescence intensity value at 480 nm, where the slope of the initial segment signified the surface hydrophobicity (S0) of HSA.
4.8. Binding Site Exploration
To determine where hispidin binds to HSA, site competition experiments were performed on the interactions between proteins, probes, and hispidin. The fluorescence titration approach was used to carry out binding location experiments between hispidin and HSA with warfarin, ibuprofen, digitoxin, and hemin. The concentration ratio of HSA and site markers was 1:3, and a full reaction was performed at room temperature for 30 min; then, hispidin was added dropwise to the site marker–HSA mixtures. A 280 nm excitation wavelength was chosen and the fluorescence emission spectra of HSA were collected.
4.9. Molecular Docking Simulation
Using Autodock 4.2, Autodock tools (ADT) software and the Lamarckian genetic algorithm were used to fit the combination of hispidin and HSA. The stereochemical structure of hispidin (CID: 54685921) and the crystal structure of HSA (PDB id: 1H9Z) were retrieved from PubChem and the Brookhaven Protein Data Bank, respectively. During the docking process, the polar hydrogen bonds and Gasteiger charges of hispidin were inserted using the AutoDock tool. A deprotonated charged state was adopted for each ligand. In order to identify potential hispidin binding sites on proteins, grid boxes of 116 Å × 126 Å × 126 Å were chosen for blind docking the search areas that encompassed the entire HSA molecule. The parameters that were set were as follows: 150, 100, 2,500,000, and 2700 for the genetic algorithm (GA) population size, number of GA runs, maximum number of energy evaluations, and maximum number of generations. Following the completion of the docking computation, Discovery Studio 4.5 software was used to examine the optimal conformation, which had the lowest binding energy.