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. 2026 Aug 18;11(34):50751–50761. doi: 10.1021/acsomega.5c13419

Investigating the Interaction between a Lipophilic Bioactive Compound and Digestive Enzymes via Interference Effects in Colloidal Crystal Films

Haixin Bian 1, Xiaonan Cao 1, Tong Zhang 1, Lele Zhou 1,*
PMCID: PMC13625102  PMID: 42819827

Abstract

Curcumin, as an example of a lipophilic bioactive compound, is typically ingested orally for absorption into the human body. However, dynamically tracking the interaction between lipophilic bioactive compounds and digestive enzymes remains a significant challenge in the study of lipophilic bioactive compounds’ absorption, translocation, and metabolism within the gastrointestinal tract. The interaction between curcumin and lipase or trypsin was monitored in real time using an ordered porous layer interference (OPLI) system based on silica colloidal crystal (SCC) films. The trypsin or lipase layers were obtained by adsorbing trypsin or lipase onto the SCC films. The optical thickness (OT) received by the OPLI system was used to monitor the interaction between curcumin and digestive enzymes in real time. By comparing the interaction processes of curcumin on trypsin layers at different pH values, it was determined that the structure of curcumin under varying pH conditions influenced its interaction with trypsin. The results for changes in optical thickness (ΔOT) showed that the ΔOT of curcumin was greater in the trypsin (24.79 ± 4.94 nm) than in the lipase (2.49 ± 0.84 nm). The combination or dissociation rate constant (K on or K off) of curcumin in the presence of the trypsin and lipase layers was compared and analyzed through ΔOT. The calculated results revealed that the affinity constant (K A) of curcumin on the trypsin layer and the lipase layer were 1.15 × 103 M–1 and 5.33 × 103 M–1, respectively. Analyzing the association and dissociation of curcumin with digestive enzymes offers real-time information and dynamic parameters for a more profound comprehension of the behavior of lipid-soluble drugs in the gastrointestinal tract.

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Introduction

Curcumin, a polyphenolic compound extracted from the rhizome of turmeric, has a wide range of biological activities, such as anti-inflammatory, antioxidant, and anticancer effects. , Investigating the interaction mechanism between curcumin-based lipid-soluble drugs and proteins is crucial for understanding their biological activity. Curcumin interacts with proteins via several mechanisms, including hydrogen bonding, hydrophobic interactions, π–π stacking, metal chelation, and covalent bonding. The distinctive molecular structure of curcumin, especially its conjugated enone-ketone system, facilitates a variety of molecular interactions with proteins. , The adsorption of curcumin to proteins may inhibit water molecule interactions, thereby protecting curcumin from hydrolytic degradation. Curcumin’s adsorption to enzymes may alter the enzyme’s function by disrupting its structure. These studies indicate that interactions between lipophilic bioactive compounds and various proteins are complex, affecting both the function of these proteins and the bioactivity of lipophilic bioactive compounds themselves.

Lipid-soluble curcumin primarily enters the body through oral administration. However, its bioavailability is low, mainly due to its poor water solubility and instability in gastrointestinal fluids. The main digestive enzymes that curcumin encounters in the gastrointestinal tract are protease and lipase. Research indicates that curcumin’s interaction with trypsin may involve both noncovalent and covalent bonds. The interaction between curcumin and trypsin is a complex and multifaceted area of study. The interactions between bovine trypsin and curcumin affect stability, structure, and function. Curcumin demonstrates various biological activities, such as anti-inflammatory, antioxidant, and antitumor effects, by inhibiting trypsin. Additionally, curcumin has a potential therapeutic role in managing obesity and diabetes by inhibiting lipase activity and regulating lipid metabolism. , The interaction between drugs and digestive enzymes is crucial for the storage, transport, and metabolism of drugs, influencing their pharmacodynamic activity. Nevertheless, the real-time dynamic monitoring of the interaction between curcumin and digestive enzymes, which is closely related to drug absorption, transport, and metabolism, has not been thoroughly studied.

Reflectometric interference spectroscopy (RIfS) is a label-free detection technique based on the principle of white light interference, primarily used to measure the thickness and surface reactions of thin films. As a label-free biosensor, RIfS technology eliminates the need for prelabeling or sample processing, which is a significant advantage in the field of biosensing for real-time, in situ detection of biomolecular interactions. − The basic principle of RIfS is that when white light is irradiated onto the film surface, the reflected and refracted light generated forms an interference pattern. When the thickness of the film changes, the position of the interference peak in the interferogram will also shift, allowing for accurate measurement of the film thickness. Fortebio in silicon valley began to study the combination of RIfS scanning technology with optical fiber technology, using it to make analytical instruments for detecting biomolecular interactions, and developed Bio-Layer Interferometry technology (BLI) for real-time monitoring of interactions between biomolecules. Surface plasmon resonance (SPR) biosensor and grating coupled intermetry (GCI) can also monitor the dynamic processes of molecular interactions in real time and with high sensitivity without the need for labeling. Compared with RIfS, SPR requires expensive gold film sensors and complex instruments, and is susceptible to temperature and other factors, resulting in higher costs. , Compared with RIfS, the technical threshold of GCI is higher, and the equipment is precise and expensive. , In contrast, the RIfS is capable of real-time detection, with simple operation, low cost, easy miniaturization, and high-throughput detection.

Constructing an interference base is the key part of the RIfS sensing method. At present, interference substrates are generally divided into two categories: one is planar solid substrates represented by inorganic oxides or polymer films, , and the other is porous silicon (PSI) − and porous anodic alumina (NAA) represented by porous substrates. Compared with the planar solid substrate, the sensitivity of the silica colloidal crystals (SCC) , porous substrate is improved to some extent due to the larger specific surface area of the porous substrate. Moreover, the porous structure of the substrate provides more space for the adjustment of the load of the material to be measured.

Unlike the columnar pores of NAA, the pores in SCC films were interconnected, which facilitated the diffusion of the measured molecule within the pores. A colloidal crystal is a two-dimensional or three-dimensional ordered array structure composed of monodisperse colloidal particles, assembled under the influence of gravity, electrostatic force, or capillary force. It had a high surface area, and its film thickness could be controlled within a few microns. Among them, the SCC films were uniform, ordered, and transparent, allowing the optical signal to be collected from the bottom of the SCC films. Therefore, even if the analyzed solution was colored or cloudy, it did not affect the acquisition of optical signals. Professor Qian Weiping’s research group studied the interference effect of SCC films and developed a series of analytical applications based on RIfS technology. The RIfS system with SCC films as the interference substrate was named the ordered porous layer interferometry (OPLI) system. ,

The study of the interaction between curcumin and proteins is the basis of explaining its various biological activities in vivo. To real-time tracking of the interaction between lipophilic compounds and digestive enzymes, an OPLI system was established based on SCC film interference migration to track the adsorption and desorption processes of curcumin on trypsin and lipase. The OPLI system translates the migration of interference peaks, caused by the interaction between proteins and curcumin, into real-time changes in optical thickness (ΔOT). The affinity parameters between curcumin and the trypsin layer or lipase layer can be calculated by the magnitude of ΔOT.

Experimental Section

Materials

Bovine serum albumin (BSA), Nα-benzoyl-dl-arginine 4-nitroanilide (BAPNA), and dimethyl sulfoxide (DMSO) were procured from Shanghai Yuanye Bio-Technology Co., Ltd., in China. Trypsin (200,000 U/g) sourced from bovine plasma was obtained from Shanghai Shifeng Biological Technology Co., Ltd., in China. Sodium hydroxide (NaOH) was purchased from Tianjin Yongda Chemical Reagents Co., Ltd. Triethyl glycerate was purchased from Tianjin Huasheng Chemical Reagents Co., Ltd. Microscope slides (76 × 26 × 1 mm3, SAIL brand, from China) were utilized as solid substrates for the growth of the SCC films. The SCC films were prepared using a glass tank (100 × 95 × 48 mm3) and a stainless steel bracket (72 × 66 × 30 mm3). Silica microspheres (200 nm) dispersed in an aqueous solution were obtained from Jiangsu Zhichuan Technology Co., Ltd. Anhydrous ethanol (EtOH), sodium hydrogen phosphate (Na2HPO4·12H2O), sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O), hydrogen peroxide, gelatin, curcumin (AR), and sulfuric acid (H2SO4) were all sourced from Sinopharm Group Chemical Reagent Co., Ltd. Water-soluble curcumin products were purchased from Shaanxi Hexin Biological Co., Ltd. By adjusting the quantities of Na2HPO4·12H2O and NaH2PO4·2H2O, PBS buffer solutions with pH values of 5.4, 6.8, 8.2, and 9.6 can be prepared. A small amount of NaOH is required when preparing PBS with a pH of 9.6. To dissolve the lipid-soluble drug curcumin, one-third of the PBS buffer solution was substituted with an ethanol solution, resulting in a PBS-ethanol (PBS + ET) solution. Lipase (100,000 U/g) was purchased from Shanghai Yusuo Chemical Technology Co., Ltd.

Preparation of SCC Films

The silica solution was replaced with ethanol as the solvent and prepared into a 0.8 wt % solution. The SCC films were prepared using the vertical growth method. First, the glass sheet was cleaned with a mixture of concentrated sulfuric acid and hydrogen peroxide. A 0.8 wt % silicon solution was poured into the dye bath. The glass sheet, placed on the dye bath stand, was vertically inserted into the silicon solution and allowed to stand at room temperature for 5 to 8 days. The SCC film, which grew to approximately 3 cm, was removed, and the silicon solution on the surface was washed with ethanol to obtain the SCC films.

Characterization of Structure and Measurement of Optical Properties of SCC Films

The morphology of the SCC films was characterized using scanning electron microscopy (EVO LS15, Zeiss) operated at either 5 or 10 kV. Reflective interference spectra were obtained through a combination of a spectrometer (FIB-600-TA-L-NIR, Idea Optics, Ltd., Shanghai, China) and an inverted optical microscope (DM2000X, Beijing Baihewei Technology Co., Ltd.). The background spectrum was recorded with air and a blank slide as the reference. The reflection interference data for SCC films were collected in the spectral range of 300–800 nm and normalized to the reflectance of the reference spectrum. Absorption spectra of curcumin solutions were measured using an LS3 visible spectrophotometer. Different solutions were injected into the porous films using a peristaltic pump, and the corresponding optical thickness (OT) was obtained online in real time. The adsorption data for curcumin on lipase and trypsin were obtained after subtracting the adsorption data for curcumin on the SCC film as a background. The adsorption of curcumin on the lipase layer and trypsin layer reflected both the interactions between curcumin and the enzymes and the interactions between curcumin and the SCC film. The OPLI system data was acquired using a peristaltic pump with a constant flow rate of 0.12 mL/min. The experiments were based on replicates from at least three independent film batches.

Assay for the Validation of Trypsin and Lipase Activity

The procedure for determining trypsin activity was as follows: first, a 20 mM BAPNA was prepared by dissolving it in DMSO, then diluted with PBS to 5 mM. A 5 mM BAPNA solution was mixed with a 1 mg/mL trypsin supernatant dissolved in PBS or PBS + ET in a 1:1 volume ratio and allowed to react at room temperature for 30 min. The absorbance was measured after a 20-fold dilution to determine the effect of ethanol addition on trypsin. After introducing 1 mg/mL trypsin supernatant through an SCC film for 60 min, the trypsin-loaded SCC film was rinsed with PBS + ET for 5 min. Subsequently, the SCC film adsorbed with trypsin was immersed in 5 mM BAPNA and reacted for 30 min. Immediately, the absorbance was measured after diluting 20-fold to determine the activity of the trypsin adsorbed onto the SCC film.

The method for determining lipase activity was as follows: 56 mg/mL triacetylglycerol with the supernatant of 1 mg/mL lipase dissolved in PBS or PBS + ET was mixed at a 1:1 volume ratio and allowed to react for 15 min. After diluting the mixture 20-fold, three parallel titrations with 40 mM NaOH were performed to determine the effect of ET addition on lipase activity. After a 1 mg/mL lipase supernatant was introduced to the SCC film for 60 min, it was rinsed with PBS + ET for 5 min. Then the lipase-loaded SCC film was immersed in 56 mg/mL triacetylglycerol and allowed to react for 30 min. The lipase activity adsorbed onto the SCC film was determined by performing three parallel titrations with 40 mM NaOH.

Results and Discussion

Characterization of SCC Films and Construction of OPLI System

In Figure , an SCC film presents a tightly packed structure over a large area. The 200 nm silica microspheres in the SCC film, acquired through vertical evaporation, display a regularly arranged hexagonal structure (Figure ). These uniform pores provide a three-dimensional space conducive to protein loading. Due to Bragg’s diffraction law, the regular structure of the SCC films causes interference when incident light coherently emitted within the SCC films. The optical path system of an inverted microscope, when combined with a reflection interference spectrum and a spectrometer, establishes the OPLI system. The adsorption and desorption of substances on the SCC films resulted in shifts of interference peaks of the SCC films. The interaction between curcumin and digestive enzymes was monitored in real time by tracking the migration of interference peaks on the SCC films using the OPLI system (Scheme ).

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(A) SEM image of SCC film. (B) Enlarged view of part of the SCC film in figure (A).

1. (A) Schematic Diagram of the Interaction between Curcumin and Digestive Enzymes; (B) Interference Effect Diagram of SCC Film; (C) Schematic Diagram of OT Variation of Curcumin Adsorption and Desorption Processes on Digestive Enzymes; (D) Schematic Diagram of the Migration of Interference Fringes.

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The effect of buffer solution on SCC films was studied before real-time tracking of the interaction between curcumin and digestive enzymes with the OPLI system. As shown in Figure A, a stable baseline can be obtained by injecting PBS into the SCC film, suggesting that the PBS buffer solution does not affect the SCC film. Similarly, Figure S1, a stable baseline, can also be obtained by injecting a PBS ethanol solution into the SCC film, indicating that the PBS ethanol solution does not affect the SCC film. The illustrations in Figures A and S1 are enlarged baselines. Curcumin, being a lipid-soluble compound, was experimentally confirmed to dissolve in a PBS solution (pH = 5.4, 6.8, 8.2, and 9.6) containing one-third ethanol (PBS + ET). As shown in Figure B, curcumin primarily exhibits yellow (Figure Ba,b) and red (Figure Bc,d) colors in solutions of different pH levels. The molecular structure of curcumin undergoes a transition from the enol form (in acidic and neutral environments) to a deprotonated form (in alkaline environments) under varying conditions. Therefore, we subsequently selected buffer solutions with pH values of 6.8 and 8.2 to dissolve curcumin, comparing the effects of curcumin’s structure on its interaction with digestive enzymes.

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(A) Real-time OT variation in SCC film with continuous PBS. (B) Photographs of curcumin in PBS + ET at different pH. (C) The absorption spectrum of the ethanol of curcumin and the ethanol of curcumin extracted from the SCC film containing curcumin. The inset of (C) was composed of photographs of SCC film and SCC film containing curcumin.

We deposited curcumin dissolved in a PBS + ET buffer solution onto the SCC film dropwise. After the solvent evaporated, the blue SCC film turns yellow in Figure C. Even after extensive washing with ultrapure water, the SCC film containing yellow curcumin retains its color (as illustrated in Figure C). This indicated that curcumin remained partially adhered to the pores of the SCC film, even under conditions of water rinsing. The ethanol solution of curcumin and the SCC film containing curcumin were then washed with ethanol for UV measurement. As shown in Figure C, both the curcumin solution on SCC film and the curcumin sample solution exhibit an absorption peak at 420 nm, attribute to aromatic phenol groups. This demonstrated the physical adsorption of curcumin by the three-dimensional porous structure of SCC films.

Real-Time Monitoring of the Interaction between Curcumin and Trypsin Layer

Curcumin was dissolved in PBS + ET buffer solution with a pH of 6.8 to study the interaction between the enol form of curcumin and trypsin. Figure A shows an example of real-time monitoring at various stages of the interaction between curcumin and trypsin using the OPLI system. The process consists of seven steps: (a) stabilizing the baseline using PBS; (b) functionalizing the surface of silica microspheres with trypsin; (c) washing with PBS; (d) rinsing with PBS + ET; (e) introducing curcumin; (f) rinsing with PBS + ET; (g) washing with PBS. After achieving a smooth baseline in PBS (pH = 6.8), we initially introduced the supernatant of 1 mg/mL trypsin into the SCC film, which resulted in an OT increase of 14.7 ± 2.5 nm. Compared to the reflectance spectra of the SCC film alone, the reflectance spectra of SCC film with trypsin adsorption are red-shifted, as depicted in Figure B. This confirmed the presence of a trypsin layer on the SCC film. After that, we passed PBS to wash off the adsorbed unstable trypsin until the OT remained unchanged. The previous PBS was replaced with a PBS + ET buffer solution and passed into the SCC film to establish a stable baseline. Then, a 0.5 mM curcumin solution, dissolved in PBS + ET buffer solution, was passed into the trypsin layer to monitor the action behavior of curcumin in the trypsin layer in real time, as shown in Figure A. Compared to the adsorption of trypsin to the SCC film, the introduction of curcumin onto the trypsin layer caused the OT in the SCC film to red-shift once again. The introduction of curcumin to the trypsin layer resulted in an OT increase of 28.3 ± 5.8 nm.

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(A) Real-time ΔOT in the interaction between curcumin and trypsin layer at pH 6.8. (B) Reflectance spectra of SCC film corresponding to (A) at different stages. (C) Linear regression curve of curcumin concentration versus ΔOT. (D) Real-time ΔOT following the introduction of curcumin (pH 6.8) into the SCC film. The adsorption fitting curve (E) and dissociation fitting curve (F) of curcumin on the trypsin layer were based on (A). (G) Real-time ΔOT of PBS + ET introduced onto the trypsin layer. (H) Absorbance values following the addition of BAPNA to the trypsin layer washed with PBS + ET, trypsin in PBS, and trypsin in PBS + ET, respectively.

To compare the ΔOT induced by curcumin on the SCC film and trypsin layer, curcumin was dissolved in a PBS + ET solution to track the real-time ΔOT of curcumin on the SCC film, as depicted in Figure D. After a stable baseline was established by flowing PBS + ET (pH = 6.8) through the SCC film, 0.5 mM curcumin was introduced into the SCC film. The introduction of 0.5 mM curcumin into the SCC film caused the OT to increase by 3.51 ± 0.86 nm. The results in Figure A,D demonstrate that curcumin introduced into the trypsin layer induces greater ΔOT than in SCC film. The ΔOT value of curcumin measured on the trypsin layer (28.3 ± 5.8 nm) was significantly higher than that measured on the SCC film (3.51 ± 0.86 nm), indicating an interaction between curcumin and trypsin. Due to the nonspecific binding of curcumin to the SCC film, after background subtraction, the adsorption capacity of curcumin to trypsin was 24.79 ± 4.94 nm. A stable baseline was obtained after flowing PBS through the SCC film. The PBS was replaced with a 1 mg/mL trypsin supernatant, as shown in Figure G. When the trypsin had adsorbed stably onto the SCC film, PBS was flowed through again. The adsorption of trypsin caused the OT of the SCC film to rise by 7.51 ± 1.91 nm. Upon replacing PBS + ET with PBS, the OT rose sharply and immediately. Finally, after replacing PBS + ET with PBS once more, the OT fell to 5.64 ± 0.52 nm. This indicates that the majority of the trypsin adsorbed following the addition of PBS + ET remained on the SCC film. These results demonstrated the interaction of curcumin with the trypsin layer.

We conducted a control experiment to investigate the effect of ethanol addition on trypsin activity. The trypsin dissolved in PBS and PBS + ET was mixed with 5 mM BAPNA in a 1:1 volume ratio and reacted for 30 min. The measured absorbance values are 1.27 ± 0.13 and 0.86 ± 0.01, respectively, as shown in Figure H. The data indicates that the addition of ET still maintains the activity of the trypsin. To investigate the activity of trypsin adsorbed onto the SCC films, it was immersed in 5 mM BAPNA and reacted for 30 min after trypsin had reached adsorption equilibrium on the SCC films and was rinsed with PBE + ET. The measured absorbance is 0.03 ± 0.02 in Figure H. This indicates that the layer of trypsin adsorbed onto the SCC film remains active. The adsorption amount is relatively small, so the activity of the enzyme on the SCC films cannot be compared with that in PBS.

The rinsing with the PBS + ET solution causes the reflectance spectrum of the SCC film to be blue-shifted (Figure B). This proved the desorption of curcumin from the trypsin layer. The OPLI system tracked the adsorption and desorption behavior of curcumin on the trypsin layer in real time. The final entry of the PBS buffer into the SCC film was consistent with the baseline environment of the initial SCC film. The PBS position of the SCC film after curcumin desorption is 3.0 nm higher compared to the initial OT of the baseline position (Figure A), indicating that the addition of PBS + ET buffer solution still retained the trypsin on the SCC film. The hydrophobic groups of curcumin can interact with the hydrophobic regions of proteins. The desorption interaction between curcumin and trypsin may be due to the interaction of the hydrophobic moiety of curcumin with the hydrophobic region of trypsin.

Curcumin molecules have hydroxyl groups at both ends, and under alkaline conditions, a conjugation effect occurs where the electron cloud deviates, leading to deprotonation of the hydroxyl groups and resulting in negative charges. We prepared curcumin using a PBS + ET solution (pH 8.2) and further analyzed its real-time interaction with trypsin. At pH 8.2, 0.5 mM of curcumin exhibits a red color (Figure A), whereas at a pH of 6.8, the same concentration of curcumin appears yellow (Figure A). After establishing a stable baseline with a PBS solution (pH 8.2), a 1 mg/mL trypsin solution was passed through the SCC film, resulting in trypsin adsorption onto the film. Loosely bound trypsin was rinsed away with PBS. Subsequently, a PBS + ET solution (pH 8.2) was passed through the SCC film to obtain a stable baseline. A 0.5 mM curcumin solution was prepared using a PBS + ET buffer (pH 8.2). This solution was then introduced into the trypsin-adsorbed SCC film to monitor the interactions between curcumin and the trypsin layer in real time. The adsorption of curcumin onto the trypsin layer resulted in a 1.9 nm OT increase. As shown in Figure B, introducing 0.5 mM curcumin (pH = 8.2) into the SCC film results in an increase in OT of 3.55 ± 0.93 nm. Curcumin carries a negative charge in alkaline solutions. Conversely, trypsin carries a positive charge in a solution with a pH of 8.2. This may be attributed to the presence of hydroxyl and methoxyl groups in curcumin, which are negatively charged in their ionized state. The electrostatic interactions between curcumin and positively charged amino acid residues, such as arginine and lysine, may not play a crucial role. Minor alterations in the OT may stem from changes in environmental pH (deprotonation), which may not only affect curcumin charge state but also induce conformational shifts. Furthermore, alterations in the solvent environment may influence the tendency for intermolecular aggregation. In comparison to the greater OT of curcumin depicted in Figure A (pH = 6.8), the lesser OT of curcumin in Figure A (pH = 8.2) is evident. Therefore, the different structures of curcumin at various pH levels resulted in a disparity in the interaction between curcumin and trypsin. Curcumin had greater adsorption to the surface of the trypsin layer in its nonionized state. In the subsequent experiments, we all used a PBS pH of 6.8. The phenolic hydroxyl groups of curcumin were protonated to form a stable nonionized form. At pH 8.2, the adsorption capacity of curcumin directly onto SCC films was greater than the adsorption capacity of curcumin on the trypsin layer. This indicates that the interaction mechanism between curcumin and trypsin may change at pH 8.2.

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(A) Real-time ΔOT in the interaction between curcumin and trypsin layers at pH 8.2. (B) Real-time ΔOT following the introduction of curcumin (pH 8.2) into the SCC film.

Analysis of the Association and Dissociation of Curcumin with Trypsin Layer

To determine the ΔOT of curcumin at various concentrations to the trypsin layer, solutions of 0.05 mM, 0.1 mM, and 0.5 mM curcumin dissolved in a PBS ethanol solution were used. A linear curve was established by plotting the curcumin concentration on the x-axis against the increase in OT caused by the introduction of curcumin onto the trypsin layer on the y-axis. As shown in Figure C, the linear relationship is described by the equation y = 49.08x + 3.76 (R 2 = 0.9998).

The association rate constant (K on) and the dissociation rate constant (K off) were determined from the curves representing the ΔOT processes of curcumin. Equations – , were used for the calculations. The use of the 1:1 Langmuir model represents a simplified approximation of the complex system involving curcumin and the digestive enzyme layer. The application of this model draws on studies focusing on the specific interactions between proteins and biomolecules using the OPLI system. The interactions between biomolecules were examined from the perspectives of antiviral activity assessment and binding mechanism analysis, respectively. ,, The affinity between curcumin and the trypsin layer was analyzed to display their kinetic characteristics. In formula , a is the termination ΔOT, and K obs is the apparent rate constant. For formula , a is the initial ΔOT. For formula , K obs links K on and Koff together. Affinity constant (K A) was determined by K on and K off (eqs ).

ΔOT=a(1−e−Kobs·t) 1
ΔOT=ae−Koff·t+b 2
Kobs=Kon[Cur]+Koff 3
KA=KonKoff 4

The process with high dissociation efficiency typically exhibits a high K on and a low K off. Using nonlinear fitting, the calculated K obs for the curcumin to the trypsin layer was 0.4917 ± 0.0139 min–1 (R 2 = 0.9737), K off was 0.3118 ± 0.0171 min–1 (R 2 = 0.8987), K on was 0.36 × 103 M–1·min–1, and K A was 1.15 × 103 M–1. The K on was high, and the K A was low. The porous structure of the SCC films may indeed introduce heterogeneous binding sites and mass transfer effects. When fitting the experimental data, we found that this model exhibited a high degree of fit for the adsorption of curcumin on the trypsin layer. The use of the 1:1 Langmuir model helped us to simplify the analysis and obtain a relatively meaningful apparent binding constant to assist in explaining the interaction between curcumin and the digestive enzyme layer. According to the K A, the affinity between curcumin and trypsin layers was relatively low.

Real-Time Monitoring of the Interaction between Curcumin and Lipase Layer

After monitoring the real-time interaction between curcumin and trypsin layer, we employed lipase, another digestive enzyme, to observe the interaction between curcumin and the lipase layer in real time using the OPLI system. An example of real-time monitoring of the interaction between curcumin and lipase layer is shown in Figure A. The OPLI system’s real-time monitoring process consists of seven steps: (a) obtaining the baseline using PBS; (b) functionalizing the surface of silica microspheres with lipase; (c) washing with PBS; (d) rinsing with PBS + ET; (e) introducing curcumin; (f) rinsing with PBS + ET; (g) washing with PBS. Upon achieving a smooth baseline in PBS, the supernatant of lipase was introduced onto the SCC film. As time progressed, lipase adsorbed onto the SCC film, resulting in an increase in OT by 14.3 ± 4.0 nm, as shown in Figure A. The red shift of the SCC film after lipase adsorption is illustrated in Figure B. Subsequently, PBS was used to wash away the loosely bonded lipase, and the OT remained relatively constant. Then, the PBS buffer solution was substituted with a PBS + ET buffer solution and passed through the SCC film to obtain a stable baseline. A 0.5 mM curcumin solution, dissolved in PBS + ET buffer solution, was then introduced into the lipase-loaded SCC film to monitor the interaction between curcumin and lipase in real time. Curcumin introduced to the lipase layer caused the SCC film to red shift again. The introduction of curcumin onto the lipase layer resulted in an increase in the OT by 6.0 ± 1.7 nm. After subtracting the background value for curcumin adsorption to the SCC film, the curcumin adsorption capacity to lipase was 2.49 ± 0.84 nm.

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(A) Real-time OT variation of curcumin adsorption and desorption behavior on lipase layer. (B) Reflectance spectra of SCC film in correspondence with (A) at different stages. (C) Real-time ΔOT of PBS + ET introduced onto the lipase layer. (D) and (E) were curve-fitting and calculation of K on and K off based on (A). (F) The concentrations of fatty acids produced by the respective reactions between lipase in the lipase layer, lipase in PBS, and lipase in PBS + ET with triacylglycerol.

The final infusion of PBS buffer was consistent with the baseline environment of the initial SCC film. At this point, the OT of the SCC film is 3.8 nm higher than that of the original SCC film (Figure A). Compared to the initial OT position, this indicates that the addition of PBS ethanol buffer solution still retained the lipase layer on the SCC film. After replacing the PBS with 1 mg/mL lipase supernatant, the OT gradually increased. Once equilibrium was reached, PBS was reintroduced. The adsorption of lipase onto the SCC film causes the OT to increase by 5.91 ± 0.82 nm, as shown in Figure C. Subsequently, when PBS was replaced with PBS + ET, the OT rose immediately and remained largely unchanged. Upon reintroduction of PBS, the OT immediately fell to 4.65 ± 1.24 nm. This indicates that lipase adsorbed onto the SCC film remained present following the introduction of PBS + ET.

A control experiment was conducted by investigating the effect of ethanol addition on lipase activity. A 1 mg/mL solution of lipase dissolved in PBS and PBS + ET was mixed with a triacetin solution in a 1:1 volume ratio and allowed to react for 15 min. The reaction mixture was diluted 20-fold and titrated with 40 mM NaOH. As shown in Figure F, the concentrations of fatty acids generated by the reaction of lipase dissolved in PBS and PBS + ET with triacetylglycerol are 240 ± 36 mM and 178.67 ± 2.31 mM, respectively. This indicates that the addition of ethanol does not inhibit lipase activity. The activity of lipase adsorbed onto the SCC film was investigated by washing the lipase-loaded SCC film with PBE + ET. It was then immersed in a triacetylglycerol solution and reacted for 40 min, the titrated fatty acid concentration was 18.67 ± 0.83 mM. This indicates that the lipase layer adsorbed onto the SCC film remains active. It was confirmed that the rise in OT of 6.0 ± 1.7 nm was caused by the curcumin action upon the lipase layer. Indeed, the addition of ethanol resulted in a decrease in the activity of both trypsin and lipase. It is important to note that ethanol may also affect the interaction between curcumin and trypsin. Nevertheless, when studying protein-curcumin interactions, an appropriate amount of organic solvent (such as DMSO or ethanol) is necessary to dissolve the hydrophobic curcumin. According to a publication, the presence of 25% ethanol induces no major BSA structural changes. Furthermore, it has been reported in the literature that the binding constant between maize alcohol-soluble protein and curcumin in 80% ethanol was determined using a fluorescence quenching method.

We further investigated the real-time interaction between curcumin and nonenzymatic proteins (gelatin) as depicted in Figure S3. After establishing a smooth baseline with PBS, a 1 mg/mL gelatin was introduced to the SCC film. Over time, gelatin adsorbed onto the SCC film, resulting in an increase in the OT by 15.3 ± 4.6 nm Subsequently, PBS was used to wash away the unstable gelatin adsorption, which caused a slight decrease in the OT. The PBS buffer solution was then replaced with a PBS + ET buffer solution, which was introduced into the SCC film to obtain a stable baseline. A 0.5 mM curcumin solution, dissolved in PBS + ET buffer solution, was then passed through the gelatin-loaded SCC film to monitor the behavior of curcumin on the gelatin layer in real time. The introduction of curcumin to the gelatin layer resulted in an increase in the OT (24.5 nm). Following the introduction of curcumin onto the gelatin layer, rinsing with PBS + ET buffer solution. The final introduction of PBS buffer aligned with the buffer environment of the initial SCC film. The PBS position was 8.7 nm higher than the initial OT position. Due to the nonspecific binding of curcumin to the films, after background subtraction, the binding amount of curcumin to gelatin was 20.99 nm. The high ΔOT values observed on the gelatin layer may indeed be related to nonspecific adsorption of curcumin. However, the ΔOT values (28.3 ± 5.8 nm) of curcumin observed on the trypsin layer were significantly higher than those observed on the lipase layer (6.0 ± 1.7 nm), despite both being hydrophobic enzymes. This suggests that this signal cannot be explained solely by nonspecific hydrophobic adsorption but rather involves interaction mechanisms specific to trypsin.

Analysis of the Association and Dissociation of Curcumin with Lipase Layer

The K on and the K off were determined by fitting the adsorption and desorption phases of curcumin and the lipase layer (Figure D,E). The K obs of curcumin to the lipase layer was 0.7145 ± 0.0317 min–1 (R 2 = 0.8398), K off was 0.1945 ± 0.0109 min–1 (R 2 = 0.8160), K on was 1.04 × 103 M–1·min–1, and K A was 5.33 × 103 M–1. The K on was observed to be high, while the K A was low. Based on the K A, the affinity between curcumin and lipase layers was considered relatively low. The binding constant for curcumin and bovine serum albumin (BSA) reported in fluorescence experiments is 3.33 ± 0.8 × 104 M–1. The K a of curcumin to zein and gliadin reported in fluorescence quenching method was 20 × 104 M–1 and 12 × 104 M–1 at 60% ethanol, respectively. The K a for curcumin binding to the glutelin, globulin, and albumin fractions was determined by tryptophan fluorescence quenching analysis to be 2.2 ± 0.11 × 104 M–1, 2.6 ± 0.03 × 104 M–1, and 1.3 ± 0.15 × 104 M–1, respectively. A combination of fluorescence spectroscopy and isothermal titration calorimetry (ITC) was employed to study the binding constant in curcumin compounds for plasma protein, which is in the range of 104–105 mol–1·kg. The K A value obtained in this study (103 M–1) is indeed 1 order of magnitude lower than the interaction between curcumin and protein observed in fluorescence studies. Fluorescence studies are typically conducted in homogeneous solutions, where curcumin comes into direct contact with proteins and undergoes specific or nonspecific binding. This study measured the interaction between curcumin and digestive enzymes adsorbed onto an SCC film. The solid-phase matrix may introduce steric hindrance, leading to reduced binding activity and consequently a lower binding constant. Although 103 M–1 represents a relatively weak affinity, such a weak interaction may actually be advantageous in drug delivery or sustained-release systems. This suggests that the interaction between the drug and the carrier may exhibit moderate reversibility, which is conducive to the sustained release of the drug at the target site and helps to avoid nonspecific retention within the body.

The advantage of this OPLI system lies in its ability to monitor in real time the interaction process between the lipid-soluble drug curcumin and the digestive enzyme layer. This method provides a robust quantitative tool for investigating the interaction behavior of polyphenolic drugs with digestive enzymes. Compared with traditional end point assays, this method allows for the continuous recording of the drug–protein interaction process, capturing drug behavior in real time. The OPLI system has been applied to real-time detection of plasmin activity in milk, chitosan hydrogel film drug release application, and kinetics and affinity analysis of the interaction between protein A and immunoglobulins G. Furthermore, it is possible to determine the duration of the interaction, which is crucial for understanding the behavior of the drug within the gastrointestinal tract.

The analysis of the K on and K off of trypsin and lipase on curcumin was investigated by the magnitude of the ΔOT (Figures A and A). As shown in Table , the ΔOT due to the adsorption of the trypsin layer (14.7 ± 2.5 nm) and the lipase layer (14.3 ± 4.0 nm) on SCC films were essentially the same. However, the rise in OT due to curcumin introduced onto the trypsin layer (28.3 ± 5.8 nm) was much greater than the OT variation due to curcumin introduced onto the lipase layer (6.0 ± 1.7 nm). The interaction process between trypsin and curcumin exhibited a higher ΔOT compared to lipase. This suggests that this signal cannot be explained solely by nonspecific hydrophobic adsorption but rather involves interaction mechanisms specific to trypsin. Trypsin is an alkaline protease with an isoelectric point of pH 10.1 and is positively charged at physiological pH 7.4. In contrast, the isoelectric point of lipase is 5.0. Lipase molecules have a relatively low number of basic amino acid residues and many acidic amino acid residues. Except for the higher ΔOT, there was not much difference in the affinity constant K A value between the two adsorption processes. It may be that although a greater quantity of curcumin is drawn toward the trypsin layer or accumulates on the surface, the interactions between them are partially loose, resulting in each individual interaction being relatively weak. It is speculated that both may bind to curcumin through hydrophobic interactions and hydrogen bonding.

1. Adsorption of Trypsin or Lipase on SCC Films, ΔOT and Kinetic Parameters of Curcumin with Trypsin Layer or Lipase Layer.

OT variation (nm) adsorption on SCC films curcumin ΔOT K on (M–1·min–1) K off (min–1) K obs (min–1) K A (M–1)
trypsin 14.7 ± 2.5 24.79 ± 4.94 0.36 × 103 0.3118 ± 0.0171 0.4917 ± 0.0139 1.15 × 103
lipase 14.3 ± 4.0 2.49 ± 0.84 1.04 × 103 0.1945 ± 0.0109 0.7145 ± 0.0317 5.33 × 103
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Conclusion

The trypsin layers and lipase layers were prepared by adsorbing trypsin or lipase onto an SCC film, respectively. The OPLI system was utilized to monitor the interaction of curcumin with trypsin layer and lipase layer in real time. The ΔOT values (24.79 ± 4.94 nm) of curcumin observed on the trypsin were significantly higher than those observed on the lipase (2.49 ± 0.84 nm), despite both being hydrophobic enzymes. The interaction process between curcumin and the trypsin layer was monitored in real time under pH conditions of 6.8 and 8.2, and the results differed. The affinity constant between curcumin and the trypsin layer was equivalent to that of the lipase layer. The effects of digestive enzymes and curcumin were compared and analyzed. Studying the interactions between lipophilic bioactive compounds and digestive enzymes helps elucidate their activity and function within the digestive tract.

Supplementary Material

ao5c13419_si_001.pdf (313.4KB, pdf)

Acknowledgments

We appreciate the financial support from the Yantai Science and Technology Innovation Development Plan (No. 2024JCYJ090), the Natural Science Foundation of Shandong Province (No. ZR2024QB407), and the National Natural Science Foundation of China (No. 22404006). We appreciate the support of Binzhou Medical School’s School Start-up Fund.

Data will be made available on request.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c13419.

  • Real-time ΔOT when PBS + ET was continuously passed into the SCC films; residual plots for the fitting of curcumin binding and dissociation on the trypsin layer and the lipase layer; real-time ΔOT of curcumin binding and dissociation on gelatin (PDF)

†.

H.B. and X.C. authors contributed equally. CRediT authorship contribution statement Haixin Bian: writing-original draft, investigation, formal analysis, data curation. Xiaonan Cao: writing-original draft. Tong Zhang: writing-original draft. Lele Zhou: writing-review and editing, visualization, supervision, methodology, investigation, conceptualization, validation, funding acquisition.

The authors declare no competing financial interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ao5c13419_si_001.pdf (313.4KB, pdf)

Data Availability Statement

Data will be made available on request.

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