Enhanced Activity of Eugenol Pickering Emulsion Using Lactoferrin‐Chlorogenic Acid Complex as a Green Stabilizer

ABSTRACT

Exploiting natural and bioactive stabilizers to fabricate green emulsions has attracted increasing attention. In this work, lactoferrin (LF) was combined with chlorogenic acid (CGA) to construct the LF‐CGA complex to prepare a eugenol (EG) Pickering emulsion (ELC/PE). The surface activity and binding mechanism of LF‐CGA were explored. Additionally, the antioxidant, antibacterial, and anti‐inflammatory capacities of ELC/PE were compared. The contact angle of LF‐CGA approached 90° (81.9°), indicating enhanced stability at the oil‐water interface. The interaction between LF and CGA was primarily driven by static quenching and hydrogen bonding. The ELC/PE consisted of 1.5 wt% LF‐CGA, with distilled water and EG as the aqueous and oil phases (15:1 v/v, aqueous/oil). The DPPH⁻, O₂ ⁻, and ABTS⁺ scavenging rates of ELC/PE were 86.3%, 75.3%, and 83.4%, respectively. The ELC/PE displayed superior antibacterial ability, along with inhibition rates of 99.0% and 99.5% against Candida albicans and Staphylococcus aureus, respectively. Additionally, the ELC/PE exhibited the most significant suppression of ear swelling (81.8%), confirming its anti‐inflammatory ability. These values were the highest among all experimental groups. The LF‐CGA complex acts as an emulsifier to enhance EG bioactivities. This novel complex holds great potential for expanding the practical applications of poorly soluble bioactive compounds.

Lactoferrin (LF) and chlorogenic acid (CGA) were used to design a green Pickering emulsion (PE) emulsifier. Several chemical methods were used to characterize the complex. The binding mechanism between LF and CGA was evaluated. The complex exhibited superior antibacterial, antioxidant, and anti‐inflammatory ability, which enhanced the eugenol PE effect. This emulsifier will widen the application of insoluble ingredients.

1. Introduction

Emulsions play a crucial role in drug delivery within the medical field to improve the solubility and bioavailability of hydrophobic drugs. Traditional emulsions require chemically synthesized surfactants for stabilization, but these materials possess limited functionality, lack biological activity, and raise potential environmental concerns [1]. Driven by the concept of green and sustainable development, replacing synthetic emulsifiers with natural, biodegradable bio‐emulsifiers to establish green emulsion systems has gained significant attention [2]. Pickering emulsions (PEs) introduce the concept of replacing surfactants with solid particles, reducing surfactant usage and aligning more closely with green, sustainable development principles. The stability of PEs is governed by the solid particle properties, oil‐to‐water ratio, and preparation conditions. In particular, the surface hydrophobicity of solid particles dominates their interfacial adsorption behavior. Particles with appropriate hydrophobicity can form a dense physical barrier to prevent droplet coalescence, endowing emulsions with excellent stability [3, 4]. Currently, a key technical challenge for PEs is to develop biologically active solid particles to stabilize the emulsion and act in concert with the loaded drugs.

Eugenol (EG), a major component of clove oil, possesses prominent antioxidant, anti‐inflammatory, and broad‐spectrum antimicrobial properties, along with favorable biocompatibility [5]. It has promising prospects in anti‐inflammatory, analgesic, infection prevention, food preservation, and pest control [6, 7, 8]. However, its insolubility limits its practical applications. Encapsulating EG in PEs can enhance its stability and bioactivity. For example, a PE exhibited 78% DPPH⁻ free radical scavenging rate after the addition of EG [9]. Another study reported EG can enhance the PE antibacterial effect, prolonging the shelf life of grapes [10]. Despite these promising results, most PEs still adopt synthetic or single‐component stabilizers with limited bioactivity. Hence, there is still an urgent need to develop green, multifunctional emulsifiers capable of cooperating with active ingredients like EG. Lactoferrin (LF) is a multifunctional green protein derived from milk along with antibacterial and anti‐inflammatory functions [11]. Studies demonstrated LF can serve as a stabilizer to encapsulate fish oil [12] and β‐carotene [13] in PEs. These designs successfully enhanced emulsion stability and entrapment efficiency, expanding the application of PEs in food systems. Nevertheless, the application of LF‐stabilized PEs in biomedical and therapeutic fields remains rarely reported. In addition, LF is high hydrophilicity and sensitive to the microenvironment, leading to poor stability at the emulsion's oil‐water interface. Recent efforts to overcome these limitations have focused on combining LF with polyphenols. For instance, LF‐epigallocatechin gallate complexes have been fabricated to stabilize PEs containing various plant oils [14]. However, compared with the vast diversity of polyphenols in nature, only a limited subset has been investigated. It is worthwhile to explore additional polyphenols with diverse bioactivities to construct LF–polyphenol complexes as green emulsifiers. This strategy would endow PEs with tailored physical and functional properties. Chlorogenic acid (CGA) is one of the most abundant and bioactive polyphenolic complexes in the daily diet. Its main functions involve antioxidant, antiviral, and antibacterial activities [15, 16, 17]. Accordingly, CGA represents a promising candidate for constructing a green PE stabilizer with enhanced biofunctions [18, 19]. Previous work has revealed the CGA modification can reduce the solubility of myofibrillar protein while improving its emulsifying capacity [20]. Similarly, the combination of LF and CGA could prevent protein aggregation, enhance foaming ability, and antioxidant activity [21, 22]. All these evidences indicate the LF‐CGA complex has great potential as a green emulsifier for PEs.

Herein, it is hypothesized LF and CGA can generate a complex as a green emulsion emulsifier to enhance the bioactivity of EG in a PE. LF endows the complex with inherent bioactivity and emulsifying capability, while CGA further increases the surface hydrophobicity, emulsifying performance and overall biofunctions of the complex. The LF‐CGA complex was fabricated and characterized. Its binding mechanism and in vitro antioxidant ability were evaluated. Furthermore, the LF‐CGA complex‐stabilized eugenol PE (ELC/PE) was prepared and characterized. Subsequently, its antioxidant, antibacterial, and anti‐inflammatory abilities were explored. The bioactivity of EG is significantly enhanced when encapsulated in ELC/PE. The LF‐CGA complex therefore holds great promise as a green emulsifier for PEs, offering a strategy to expand the application of unstable and poorly soluble ingredients.

2. Results and Discussion

2.1. Preparation of LF‐CGA Complex

The desired molecular concentration ratio between LF and CGA to generate LF‐CGA complex was optimized and the corresponding results are presented in Figure 1A. As the molar ratio of LF to CGA increased from 1:5 to 1:10, the particle size of the LF‐CGA complex increased obviously. It is worth noting the average particle size exhibited obvious change as molecular concentration ratio of LF to CGA kept increasing from 1:10 to 1:80. The minimal particle size (281.71 ± 15.28 nm) was obtained at the molar ratio of 1:20, and no significant improvement in polyphenol binding capacity was observed when the CGA ratio was further elevated. Emulsifiers with smaller particle sizes can migrate and adsorb onto the oil‐water interface more rapidly, achieving superior emulsifying performance. Accordingly, the molar ratio of 1:20 was selected as the optimal condition for subsequent experiments.

FIGURE 1.

The characterization of the LF‐CGA complex. (A) Polyphenol binding capacity and particle size; (B) Particle size and Zeta potential; (C) Fluorescence spectra; (D) FTIR spectra; (E) SEM image of LF. (F) SEM image of LF‐CGA. Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01.

2.2. Characterization of LF‐CGA Complex

2.2.1. Particle Sizes and Zeta Potential

The average particle size of the optimized LF‐CGA complex was 278.87 ± 8.11 nm, which was significantly larger than that of LF (176.97 ± 7.93 nm). The LF and LF‐CGA exhibited comparable polydispersity index (PDI) with 0.259 ± 0.0135 and 0.292 ± 0.01, respectively (Figure 1B). This observation can be attributed to the binding of CGA and LF with the help of intermolecular forces. The zeta potential of the LF was (7.7 ± 0.352) mV, which was consistent with previous research (8.0 ∼ 8.5) [23]. The Zeta potential of LF‐CGA was (6.2 ± 0.27) mV, which displayed a significant decrease compared to that of LF (p < 0.05). The decrease in zeta potential may be attributed to the adsorption of negatively charged CGA molecules onto the LF surface, which partially shielded the positive charges of LF. The results confirmed the formation of LF‐CGA complex.

2.2.2. Fluorescence Spectra

Fluorescence spectra is shown in Figure 1C. LF has the maximum fluorescence intensity close to 332 nm wavelength ascribed to tryptophan residues. The LF‐CGA complex showed a fluorescence intensity of 11.43 a.u., and its maximum emission peak underwent a 35 nm redshift relative to LF (196.24 a.u). This spectral change was attributed to the unfolding of the LF protein structure and greater exposure to the tryptophan residues after the binding with CGA. Moreover, the interaction between CGA and the tryptophan residues in LF also led to a decrease in fluorescence intensity [24]. These findings provide further evidence for the successful interaction between LF and CGA.

2.2.3. Fourier Transform Infrared (FTIR) Spectra

FTIR spectra of LF and the LF‐CGA complex are displayed in Figure 1D. For the LF‐CGA complex, the characteristic absorption band at 3300 cm⁻¹ corresponded to O‐H and N‐H stretching vibration and hydrogen bonding. This indicated the amino groups (─NH₂) of LF were involved in CGA binding. In addition, these hydrogen bonds may also contribute to the high stability of the LF‐CGA complex. The amide I band at the range of 1680–1630 cm⁻¹ was attributed to the stretching vibration of C═O and COO⁻ groups. Compared to LF, the amide I band of the LF‐CGA complex underwent a redshift of 1.86 cm⁻¹, suggesting a decrease in the force constant of C═O stretching vibration. This change indicated the secondary structure change of LF, and the carbonyl groups might participated in the enhanced hydrogen bonds. Additionally, the amide II band of the LF‐CGA complex underwent a redshift of 1.09 cm⁻¹, which was related to the C─N stretching vibration and N─H bending vibration. This spectral shift suggested that C─N bond interactions also participated in the binding between LF and CGA. These results are also align with existed studies [25, 26].

2.2.4. Scanning Electron Microscopy (SEM) Image

The micromorphologies of LF and the LF‐CGA complex are shown in Figure 1E,F. LF exhibited a relatively smooth spherical morphology. In contrast, the LF‐CGA complex presented an obvious lamellar structure. This morphological difference confirmed the interaction between LF and CGA. This result further validated the fluorescence spectra. It was attributed to the unfolding of LF polypeptide chains [27]. Previous research has demonstrated polyphenols disrupted the intermolecular forces and induced loose structural arrangements [28]. This research is in agreement with the above report, further verifying the formation of the LF‐CGA complex and the CGA binding induces structural rearrangement of LF.

2.3. Emulsification Property

The emulsification ability of the LF‐CGA complex for EG emulsions is indicated in Figure 2A. The centrifugal stability constant (K _e) was employed to assess emulsion stability, where a lower K _e value represents better stability, emulsification property, and homogeneous droplet distribution [29, 30]. It was calculated through Equation (1) mentioned in Section 4.4.1. The K _e of LF and LF‐CGA were 26.37 and 12.24, respectively. The result implied the LF‐CGA complex possessed superior emulsifying capacity and could effectively stabilize EG PEs.

FIGURE 2.

Properties of LF‐CGA Complex. (A) Emulsification property; (B) Fluorescence intensity; (C) Water contact angle; (D) Heat stability. Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01.

2.4. Surface Hydrophobicity

The surface hydrophobicity results of LF and LF‐CGA complex are illustrated in Figure 2B. The fluorescence intensity of LF‐CGA was significantly reduced in comparison to that of LF, indicating the hydrophobicity of LF‐CGA was increased. This change can be explained by the structural rearrangement of LF. During complexation, LF underwent conformational unfolding and internal hydrophobic groups were exposed [31]. Water contact angle measurements are presented in Figure 2C. The contact angles of LF‐CGA was 81.9°, while LF was 72.8°. The contact angle of the LF‐CGA complex was closer to 90°, a typical characteristic of ideal Pickering stabilizers with favorable emulsifying properties [32, 33].

2.5. Heat Stability

The thermal stability of the LF‐CGA complex is displayed in Figure 2D. The denaturation temperature (T _d) of LF and LF‐CGA was 65.17°C and 77.83°C, respectively. The result indicated the thermal stability of LF increased, accompanied by the formation of the LF‐CGA complex. This improvement was mainly attributed to the hydrogen bond between LF and CGA. These bonds promoted a more ordered protein structure and inhibited the aggregation of LF molecules [34]. This observation is consistent with the findings of earlier research [35].

2.6. Binding Mechanism of LF‐CGA Complex

CD spectra was used to determine the LF secondary structure modifications, thereby further analyzing the binding mechanism between LF and CGA. UV and fluorescence spectra were further adopted to monitor the variations in LF tertiary structure and explain the reasons for secondary structural transformation [36]. Combined with thermodynamic analysis and molecular docking simulations, we identified the non‐covalent interactions between the two molecules. All results established a clear correlation between molecular interactions and the functional performances of the LF‐CGA complex.

2.6.1. Circular Dichroism (CD) Spectra

The CD spectra of LF and CGA are presented in Figure 3A. A remarkable reduction in the absorption peak at 200–220 nm was observed for the complex, proving CGA binding induced changes in the secondary structure of LF. As shown in Figure 3B, LF consisted of 20.52% α‐helix (H), 32.53% β‐sheet (S), 21.26% β‐turn (T), and 25.69% random coil (C) in its secondary structure. After complexation with CGA, the relative contents changed to 16.52% α‐helix, 33.06% β‐sheet, 23.56% β‐turn, and 26.81% random coil. Notably, the LF‐CGA complex exhibited a decrease in α‐helix along with an increase in β‐turn and random coil (p < 0.01), indicating that CGA may have a protein unfolding effect on the structure of LF. This structural alteration was ascribed to the breakage of intrinsic hydrogen bonds between carbonyl oxygen and amino hydrogen in LF. Such changes lead to the improved surface hydrophobicity of LF and emulsifying stability of the LF‐CGA complex [37]. These structural variations also accounted for the changes in FTIR spectra, surface hydrophobicity and contact angle.

FIGURE 3.

The binding mechanism between LF and CGA. (A) CD spectra; (B) Secondary structure content of proteins determined by CD spectra; (C) UV spectra; (D) Intrinsic fluorescence spectra. Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01.

2.6.2. Ultraviolet (UV) Spectra

The UV spectra of the LF‐CGA complex are shown in Figure 3C. The absorption peak at 325 nm originated from CGA. The intensity of this peak rose gradually as the concentration of CGA increased. The maximum absorption of LF was red‐shifted from 282 to 299 nm during the formation of the LF‐CGA complex. This may be caused by the interaction between the hydrophobic groups in CGA and the aromatic amino acid residues (e.g., tyrosine and tryptophan) in LF [25]. K_app denotes the apparent binding constant between LF and CGA. The K_app of the LF‐CGA complex was 6.29 × 10⁴ L/mol. These values were consistent with the typical range of K_app in other protein‐polyphenol complexes [38]. The favorable binding affinity suggested the LF‐CGA complex could be stable to withstand emulsion preparation and storage without undergoing aggregation [39].

2.6.3. Intrinsic Fluorescence Spectra

As presented in Figure 3D, LF exhibited a maximum fluorescence emission at 334 nm, while the peak redshifted to 359 nm after forming the LF‐CGA complex. In addition, the fluorescence intensity of the LF‐CGA complex decreased significantly relative to LF. This finding revealed that CGA altered the tertiary structure of LF during complex formation. Obvious fluorescence quenching was detected for the LF‐CGA complex, demonstrating CGA exerted a quenching effect on LF. This phenomenon further proved LF formed a noncovalent complex with CGA [40]. The redshift of maximum fluorescence suggested the addition of CGA may result in the induced the unfolding of LF conformation [41]. Such structural changes raised the polarity of the microenvironment around LF, which further supported the surface hydrophobicity, contact angle, emulsion stability, CD and UV spectra results of the LF‐CGA complex [42].

The Stern–Volmer equation was applied to evaluate the fluorescence quenching mechanism between LF and CGA, and the results are shown in Figure 4A. K _q refers to the molecular quenching rate constant. The maximum value of biomolecules was known to be 2.0 × 10¹⁰ s⁻¹ mol⁻¹·L [43]. A K _q below this value corresponds to dynamic quenching, while a higher value indicates static quenching. Dynamic quenching arises from transient molecular collisions followed by immediate separation. By contrast, static quenching is caused by the formation of non‐fluorescent ground‐state complexes, which hardly dissociate under normal conditions [44]. The K _q values were 1.19 × 10¹³, 1.11 × 10¹³, and 1.02 × 10¹³ L·s⁻¹·mol⁻¹· at the temperatures of 298, 304, and 310 K. All these values far exceeded the maximum dynamic quenching rate constant of biological macromolecules. These results confirmed the interaction between LF and CGA was static quenching [45]. The quenching constants K_SV were 1.19 × 10⁵, 1.11 × 10⁵, and 1.02 × 10⁵ mol⁻¹·L at the temperatures of 298, 304, and 310 K. The K_SV values decreased with increasing temperature. This trend suggested the elevated temperatures weaken the structural stability of the LF‐CGA complex. This further verified that the LF‐CGA interaction belonged to static quenching, and the formed complex was a stable non‐fluorescent ground‐state complex.

FIGURE 4.

Binding analysis and emulsion characterization. (A) Stern–Volmer fitting; (B) Static quenching fitting; (C) Calibrated Stern–Volmer fitting; (D) Molecular docking analysis; (E) Interaction between LF and CGA in molecular docking; (F) Average size and Zeta potential. Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01.

As the Stern–Volmer equation was more suitable for analyzing the dynamic quenching, the Modified Stern–Volmer equation was used to calculate the accurate binding constant between LF and CGA according to Equation (4) [46]. The linear regression of F₀/$\mathrm{\Delta }\mathrm{F}$ versus 1/[CGA] is illustrated in Figure 4B. The calculated effective binding constant (K _a) decreased with increasing temperature. The K _a values were 1.24 × 10⁴, 0.53 × 10⁴, and 0.16 × 10⁴·mol⁻¹·L at the temperatures of 298, 304, and 310 K, respectively. The trend was consistent with the results of K _q, K _sv obtained from the standard Stern–Volmer analysis, which reconfirmed the stronger binding of CGA and LF at 298 K.

K _b represents the binding constant for static quenching between LF and CGA. This parameter was calculated through Equation (5) and presented in Figure 4C. The higher values of K _b indicated the stronger binding ability [43]. The K _b values in Equation (5) were 1.45 × 10⁸, 1.12 × 10⁸, and 0.48 × 10⁸ mol⁻¹·L at the temperatures of 298, 304, and 310 K. Together with the K _q and K_sv, the findings further supported the LF‐CGA complex possessed excellent binding affinity. Such strong binding enables the complex to stably adsorb at the oil‐water interface and maintain emulsion stability. The parameter n in Equation (5) stands for the binding sites per LF molecule for CGA. The corresponding n values at 298, 304 and 310 K were 1.33, 1.35 and 1.22, respectively, indicating there was one high‐affinity binding site between LF and CGA.

2.6.4. Thermodynamic Results

The thermodynamic parameters, including enthalpy ($\mathrm{\Delta }H$), entropy ($\mathrm{\Delta }S$), and Gibbs free energy ($\mathrm{\Delta }G$) were used to analyze the noncovalent interactions between LF and CGA [36]. When $\mathrm{\Delta }H$ > 0 and $\mathrm{\Delta }S$ > 0, the intermolecular binding is dominated by hydrophobic forces. For systems with $\mathrm{\Delta }H$ < 0 and $\mathrm{\Delta }S$ < 0, the intermolecular binding are mainly driven by hydrogen bonds and van der Waals forces. If $\mathrm{\Delta }H$ < 0 and $\mathrm{\Delta }S$ > 0, electrostatic interactions become the dominant driving force [36]. The $\mathrm{\Delta }H$ and $\mathrm{\Delta }S$ between LF and CGA were −132.98 and −367.23 J·mol⁻¹·K⁻¹, respectively. The $\mathrm{\Delta }G$ at 298, 304, and 310 K were −23.62, −21.41, and −19.21 kJ·mol⁻¹, respectively. Accordingly, the binding affinity of the LF‐CGA complex was dominated by hydrogen bonds and van der Waals forces. Recent research has reported non‐covalent interactions between polyphenols and proteins can enhance protein adsorption at the oil‐water interface, thereby improving emulsion stability [39]. Consistent with this, the LF‐CGA complex acts as an eligible stabilizer for PEs. This outcome further validates the results from CD, UV, and intrinsic fluorescence analyses. Moreover, these noncovalent interactions are reversible and may dissociate under specific conditions, enabling the release of LF and CGA while preserving their original bioactivity [47].

2.6.5. Molecular Docking

Molecular docking results of LF and CGA obtained via AutoDock are displayed in Figure 4D,E. The simulation revealed hydrogen bonds formed between CGA and multiple amino acid residues of LF, including PHE‐190, GLY‐194, TYR‐192, and ASP‐297. Hydrogen bonds with distances less than 3.0 Å are defined as strong intermolecular interactions [48]. The corresponding bond lengths were 2.3, 2.7, 2.6, and 2.4 Å, respectively. These data confirmed strong hydrogen bonds between LF and CGA, contributing to structural stability and favorable emulsifying capacity. These findings also supported the conclusions drawn from previous spectroscopic analyses. Apart from hydrogen bonds, hydrophobic interactions were also observed at the sites of ASP‐60, LEU‐59, MET‐63, and ALA‐42 between LF and CGA. This further explains the increase in surface hydrophobicity of the LF‐CGA complex. The predicted binding energy of the LF‐CGA complex was ‐9.4 kJ. Notably, the exposed aromatic residues such as TYR‐192 and PHE‐190 may enhance the π interactions with free radicals and cell membranes for bacteria and fungi. These changes could attribute to enhancing the LF‐CGA complex biofunctions [49].

2.7. Particle Size and Zeta Potential of ELC/PE

The Zeta potential and particle size are depicted in Figure 4F. Replacing LF with the LF‐CGA complex as the PE stabilizer greatly reduced the particle size (p < 0.01). This phenomenon could be explained by the superior emulsifying capacity of the LF‐CGA complex. ELC/PE Pickering emulsion had lower Zeta potential (p < 0.05). This change may resulted from the charge neutralization effect between LF and CGA. According to Stokes' law, smaller droplets are less likely to coalesce. Smaller droplets indicates stronger interfacial adsorption and a more robust steric barrier, leading to greater emulsion stability [50]. Hydrophobic analysis confirmed the LF‐CGA complex exhibited a contact angle closer to 90° and higher surface hydrophobicity. Together with the smaller droplet size of ELC/PE, these results further confirmed the LF‐CGA complex possessed enhanced steric hindrance. Such steric barriers effectively prevent droplet coalescence and thus form stable EG PEs. These findings also aligned with the results above, proving the LF‐CGA complex was an ideal stabilizer for PEs.

2.8. EG Content and Emulsion Type of ELC/PE

ELC/PE was identified as an oil‐in‐water (O/W) emulsion (Figure S1). High‐performance liquid chromatography (HPLC) was used to measure EG content in PEs. The EG concentration of LF‐stabilized and LF‐CGA‐stabilized emulsions were 61.93 ± 10.23 mg/mL and 62.48 ± 7.19 mg/mL, respectively.

2.9. Antioxidant Ability of ELC/PE In Vitro

1,1‐Diphenyl‐2‐picrylhydrazyl (DPPH⁻), Superoxide anion radical (O₂ ⁻), 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS⁺), and ferric ion (Fe³⁺) are essential free radicals of oxidative stress in humans and are widely present in various inflammatory diseases [51, 52, 53]. As a green Pickering emulsifier, LF‐CGA complex's DPPH⁻ radical scavenging, O₂ ⁻ radical scavenging, ABTS⁺ radical scavenging, Fe³⁺ reducing ability were initially determined (Figure 5A,B). The DPPH⁻ scavenging rate of the LF‐CGA complex was 91.38%, which was significantly higher than that of LF. Its O₂ ⁻ radical scavenging rate was 70.81%, which was over 6‐fold higher than that of LF. The ABTS⁺ scavenging capacity of the LF‐CGA complex was equivalent to 94.59 µg Trolox per milligram, while LF only showed 10.84 µg Trolox per milligram. For Fe³⁺ reduction, the LF‐CGA complex was 80.15 µg Trolox/mg and was significantly higher than that of LF (4.08 µg Trolox/mg).

FIGURE 5.

The in vitro oxidant activity of LF‐CGA complex. (A) The scavenging capacity of DPPH⁻ and O₂ ⁻; (B) The scavenging capacity of ABTS⁺ and Fe³⁺. Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01.

Taken together, the in vitro antioxidant activity of the LF‐CGA complex was significantly higher than that of LF (p < 0.01). This finding was consistent with previous reports that protein‐polyphenol conjugates improve the antioxidant performance of proteins [54, 55, 56, 57]. This may be due to the excellent antioxidant activity of CGA, offered by its phenolic hydroxyl groups. Additionally, as revealed in binding mechanism analysis, CGA binding induced the exposure of aromatic residues in LF. These residues acted as hydrogen donors to scavenge free radicals [58]. Moreover, previous studies have shown CGA binds to LF at a specific cavity [49]. The reports agreed with the molecular docking results. Such interaction shielded the phenolic hydroxyl groups of CGA and protected them against oxidative degradation. This effect maintained its radical scavenging ability and further enhanced the overall antioxidant activity of the LF‐CGA complex.

The LF‐CGA complex with antioxidant properties was adopted to prepare PEs. The free radical scavenging rates are shown in Figure 6A–C. The DPPH⁻, O₂ ⁻, and ABTS⁺ scavenging rates of LF/PE and LC/PE were 4.96% versus 18.42%, 5.83% versus 22.14% and 10.16% versus 13.12%, respectively. The three scavenging rates of LC/PE were obviously higher than that of LF/PE (p < 0.05), demonstrating the LF‐CGA complex could facilitate the antioxidant activity. EG presented a stronger free radical scavenging ability against DPPH⁻, O₂ ⁻, and ABTS⁺ with corresponding rates of 37.37%, 46.98%, and 62.12%, respectively. The antioxidant activity was further boosted as EG was encapsulated into PEs (p < 0.01), and ELC/PE was significantly higher than that of ELF/PE (p < 0.05 or p < 0.01). LF, CGA and EG exert antioxidant functions via metal ion chelation and direct radical scavenging, which rely on their phenolic hydroxyl groups. The results from surface hydrophobicity and binding mechanism analysis showed the LF‐CGA complex is an ideal emulsifier capable of stable adsorption at the oil‐water interface. This leads to a uniform PE, protecting EG from oxidative degradation and maintaining its antioxidant activity.

FIGURE 6.

Antioxidant and antimicrobial activities of ELC/PE in vitro. (A) DPPH⁻ free radical scavenging activity; (B) O₂ ⁻ free radical scavenging activity; (C) ABTS⁺ free radical scavenging activity; (D) Inhibition rate against Candida albicans; (E) Inhibition rate against Staphylococcus aureus. Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01.

2.10. Antibacterial Ability of ELC/PE In Vitro

Candida albicans (C. albicans) and Staphylococcus aureus (S. aureus) were chosen as representatives of fungi and bacteria. All samples were assigned into seven groups: control (CON), olive oil (OO), LF‐stabilized blank emulsion (LF/PE), LF‐CGA‐stabilized blank emulsion (LC/PE), EG, LF‐stabilized EG PE (ELF/PE) and LF‐CGA‐stabilized EG PE (ELC/PE). The inhibition rates are shown in Figure 6D,E. The inhibition rates of LF/PE against C. albicans and S. aureus were 14.18% and 16.28%, while those of LC/PE reached 37.54% and 24.6%, respectively. LF/PE showed comparable inhibition against C. albicans and S. aureus. In contrast, LC/PE exhibited a distinct discrepancy in antibacterial activity. This phenomenon occurred because CGA exerted stronger antibacterial activity against S. aureus than C. albicans [59]. The results were significantly higher than those of CON (p < 0.01). EG achieved inhibition rates of 59.21% against C. albicans and 62.27% against S. aureus, which were much higher than LF/PE and LC/PE. It suggested EG had a stronger inhibitory efficiency than those of LF/PE and LC/PE. The ELF/PE against C. albicans and S. aureus was increased to 92.14% and 92.74%, respectively. ELC/PE further increased the inhibition rates to 99.01% for C. albicans and 99.57% for S. aureus. It implied the addition of EG significantly improved the inhibitory effect (p < 0.01). ELC/PE presented outstanding antibacterial performance, and its activity trend was consistent with the antioxidant results. This was due to LF, CGA, and EG all possessing the ability to disrupt bacterial and fungal biofilms. As revealed by CD spectra, UV spectra, and molecular docking analyses, the conformation of LF was rearranged, and more aromatic residues were exposed. This change promoted electrostatic interactions between the LF‐CGA complex and microbial cell membranes, enhancing antibacterial activity [60]. Additionally, similar to antioxidant analysis, the LF successfully protected the bioactivity of CGA. The LF‐CGA complex also acted as an excellent carrier for EG encapsulation. All the above factors collectively endowed ELC/PE with prominent antibacterial activity.

2.11. Anti‐Inflammatory Ability of ELC/PE In Vivo

The ear edema model was established in Kunming (KM) mice to evaluate the anti‐inflammatory activity in vivo (Figure 7A–C). As shown in Figure 7B, the ear edema weight of the model control group (MC) was significantly higher than that of the normal control group (NC). These results confirmed the ear edema model was successfully constructed. All treatment groups presented obvious reductions in comparison to MC (p < 0.05 or 0.01). No visible difference among OO, LF/PE, and LC/PE was seen. The ear edema weights of EG, ELF/PE, and ELC/PE were markedly lessened than the above three groups (p < 0.05 or p < 0.01). Figure 7C displays the edema inhibition rates. The values were 53.93% for EG, 68.98% for ELF/PE and 81.81% for ELC/PE (p < 0.05). This indicated the addition of EG could alleviate ear edema and enhance anti‐inflammatory performance. ELC/PE exerted a much stronger effect than ELF/PE (p < 0.05) and similar to positive control group (PC). This finding revealed the LF‐CGA complex greatly enhanced the anti‐inflammatory effect. This trend was consistent with the antibacterial experiment.

FIGURE 7.

In vivo anti‐inflammatory ability of the Pickering emulsion (A) The establishment of the mice ear edema model; (B) The mice ear swelling weight; (C) The mice ear inhibition rate. Data are presented as mean ± SD (n = 4). *p < 0.05, **p < 0.01.

Superoxide dismutase (SOD) and myeloperoxidase (MPO) are typical endogenous enzymes in cells. The surge in reactive oxygen species (ROS) levels stimulated by pathogens causes oxidative stress, which can be reflected by the reduction of SOD levels and the increase of MPO levels [61]. High ROS levels can activate inflammatory pathways and damage cells, thereby promoting inflammation. The result of SOD levels are shown in Figure 8A. All treatment groups exhibited remarkable rose in SOD activity compared with the MC group (p < 0.05 or p < 0.01). The activity of SOD enzyme in LF/PE and LC/PE was increased, which indicated LC/PE had stronger anti‐inflammatory performance than that of LF/PE (p < 0.05). The result was attributed to the anti‐inflammatory ability of CGA. The ELF/PE and ELC/PE increased the SOD activities up to 57.72 and 59.91 U/mL, which approached the level of positive control. The ELC/PE exhibited the best effect, proving LF‐CGA complex and EG played an additive effect.

FIGURE 8.

Anti‐inflammatory ability of EG Pickering emulsions in vivo. (A) SOD activity; (B) MPO activity; (C) NO content.

SOD level is negatively correlated with MPO content [62]. The results of MPO levels are shown in Figure 8B. In comparison to MC, all treatment groups presented obvious reductions (p < 0.05 or 0.01). MPO activities of LF/PE and LC/PE dropped significantly (p < 0.01), owing to the antioxidant properties of LF and CGA. Notably, MPO level in LC/PE was much lower than that in LF/PE. The result further verified the addition of CGA remarkably strengthened the antioxidant ability. The MPO level of ELF/PE was 1.25 U/g, which was significantly reduced compared to that of EG (p < 0.01). Among all groups, ELC/PE exhibited superior effect, which was due to the combined anti‐inflammatory ability of EG and LF‐CGA complex.

Nitric oxide (NO) can react with ROS to form reactive nitrogen species (RNS). It is involved in the regulation of oxidative stress and inflammatory responses [63]. Elevated NO content usually indicates severe inflammation [64]. The result of NO levels are shown in Figure 8C. All treatment groups caused a significant decrease in NO level (p < 0.05) up to the normal level of NO. The NO level was significantly decreased in LF/PE and LC/PE (p < 0.01), which was due to the anti‐inflammation ability of LF and CGA. Interestingly, ELC/PE further lowered NO content, indicating the LF‐CGA‐stabilized EG PE possessed stronger anti‐inflammatory activity. Overall, ELC/PE exerted excellent anti‐inflammatory activity. This was attributed to LF, CGA, and EG possessing intrinsic anti‐inflammatory abilities. In addition, consistent with the antioxidant test results, ELC/PE had outstanding antioxidant capacity. This would contribute to limiting the synthesis of pro‐inflammatory mediators, thereby alleviating inflammation [65]. Moreover, as reported in antioxidant and antibacterial ability, the LF‐CGA complex successfully encapsulated EG in the PE. This system enabled EG to fully exert its anti‐inflammatory effects.

Collectively, the LF‐CGA complex effectively enhanced the functional properties of the EG PE, making it an excellent green emulsifier for broad application prospects.

3. Conclusions

In this study, a novel green PE stabilizer, the LF‐CGA complex, was successfully fabricated. The complex can achieve drug‐excipient unification, focusing on emulsifier function and enhancing bioactivities of EG PE. Spectroscopic and molecular docking analyses verified the LF underwent obvious conformational rearrangement upon binding to CGA. The binding mechanism involved hydrogen bonds and van der Waals forces. Notably, these changes could enhance the stability and emulsification performance of the complex. In addition, the LF‐CGA complex exhibited greater antioxidant performance compared with LF. More importantly, the bioactivities of the EG PE were remarkably improved after stabilized by the LF‐CGA complex. The LF‐CGA complex successfully protected EG from degradation and acted in additive with it, providing enhanced antioxidant, antibacterial and anti‐inflammatory activities. These results demonstrated the LF‐CGA complex is a promising green stabilizer integrating emulsifying performance and inherent bioactivity. Despite these promising results, the antioxidant, antibacterial, and anti‐inflammatory mechanisms of the LF‐CGA complex still need subsequent evaluation. Furthermore, the application of the LF‐CGA complex in the medical field also requires assessment.

4. Experimental Section

4.1. Materials

LF (purity: 95%), CGA (purity: 98%), and EG (purity: 98.5%) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Pyrogallol was obtained from Wuhan Kemik Biomedical Technology Co., Ltd. 1,1‐Diphenyl‐2‐picrylhydrazyl (DPPH), 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) and trichloroacetic acid were supplied by Aladdin Biotechnology Co., Ltd. Methylene blue and Sudan III were purchased from Sigma‐Aldrich, USA. Dexamethasone acetate cream was provided by Guangdong Hengjian Pharmaceutical Co., Ltd., and olive oil was acquired from Zhejiang Wumi Biotechnology Co., Ltd.

Male Kunming mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd., China. All the experiment procedures were conducted according to the protocol approved by the Institutional Animal Care and Use Committee of Jilin University School of Pharmaceutical Science (Number of permit: 20220080).

4.2. Preparation of the LF‐CGA Complex

The preparation of the LF‐CGA complex was performed as previously described [49]. Aqueous solutions of CGA and LF (1.5 mmol/L for both) were mixed at a series of molar ratios (1:5, 1:10, 1:20, 1:40, and 1:80) under stirring conditions (120 rpm). The mixture was stirred at 120 rpm for 6 h at 25°C. Then the reaction solution was dialyzed against distilled water for 24 h and freeze‐dried to yield the LF‐CGA complex. A lyophilized LF powder without CGA was prepared via the same procedure as the blank control.

4.3. Characterization of the LF‐CGA Complex

4.3.1. Particle Size and Zeta Potential

The particle size and Zeta potential of LF‐CGA solution at the concentration of 1.5 mM/L were measured on a nanolaser particle size analyzer (Shanghai Spectris Instruments System Co., Ltd., Nano ZS, China) [66].

4.3.2. Fluorescence Spectra

Fluorescence measurements were performed using a fluorescence spectrometer (Shimadzu Corporation, RF‐5301, Japan) at 25°C. The excitation wavelength was set to 280 nm, with excitation and emission slit widths of 3.0 nm and 5.0 nm, respectively [67].

4.3.3. Fourier Transform Infrared (FTIR) Spectra

FTIR spectra were recorded using a Fourier transform infrared spectrometer (PerkinElmer, USA) at the wavelength range of 400–4000 cm⁻¹ [37].

4.3.4. Scanning Electron Microscopic (SEM) Images

The SEM analysis was performed following previous reports with slight modifications [37]. The micromorphology of the LF‐CGA complex was observed by SEM. Sample powders were spread on conductive adhesive, and excess powder was gently removed. All samples were gold‐sputtered and equilibrated for 30 s before imaging. Micrographs were captured at an accelerating voltage of 5 kV with magnifications of 1000×.

4.4. Properties of LF‐CGA Complex

4.4.1. Emulsification Property

LF and LF‐CGA lyophilized powders (0.15 g each) were separately dispersed in 9.38 g of distilled water to prepare the aqueous phase. EG (0.62 mL) was added dropwise to the aqueous phase and stirred at 500 rpm for 0.5 h to form PEs. The emulsions were centrifuged at 4000 rpm for 15 min, and the absorbance was measured at 500 nm. The centrifugal stability constant (K _e) was evaluated through Equation (1) [68].

$${\mathrm{K}}_{\mathrm{e}}\left(\%\right)=[\left({\mathrm{A}}_0-{\mathrm{A}}_1\right)/{\mathrm{A}}_0]\times 100$$ (1)

where A₀ represents the absorptance of the PE before centrifugation, A₁ represents the absorptance of the PE after centrifugation.

4.4.2. Hydrophobicity Property

Surface hydrophobicity was characterized using the 8‐anilino‐1‐naphthalenesulfonic acid (ANS) fluorescence probe. LF‐CGA solution was diluted with distilled water to a concentration of 40 µg/mL. ANS solution (40 µL, 8 mM) was mixed with 4 mL of the diluted sample and incubated for 1 min. The excitation wavelength was set at 390 nm. The fluorescence intensities were collected on a fluorescence spectrometer (Shimadzu, RF‐5301, Japan) at the emission wavelength 470 nm with excitation and emission slit width of 5 nm [69]. A sample solution without ANS was used as the blank control.

Additionally, the water contact angle was measured on an optical contact angle measuring device (LAUDA Scientific GmbH, LSA100, Germany). Samples were mounted on glass slides and allowed to reach equilibrium. Droplet profiles were captured and analyzed by fitting to the Laplace–Young equation.

4.4.3. Thermal Stability

Thermal stability was analyzed using a thermogravimetric analyzer (NETZSCH, Germany). In brief, LF and LF‐CGA powders were weighed, sealed and kept at room temperature for 6 h prior to testing. Measurements were carried out over the temperature range of 50–180°C under a nitrogen flow of 30 mL/min [70].

4.5. Binding Mechanism of LF‐CGA Complex

4.5.1. Circular Dichroism (CD) Spectra

The CD spectra was performed following previous reports with slight modifications [37]. 0.1 mL of aqueous LF‐CGA solution (0.4 mg/mL) and 0.1 mL of aqueous LF solution (0.4 mg/mL) were mixed to scan in the range of 180–260 nm wavelength to obtain CD spectra.

4.5.2. Ultraviolet (UV) Spectra

LF solution (1 mL, 4 mg/mL) was blended with 0.1 mmol/L CGA solution (0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mL). Distilled water was supplemented to adjust the total volume of each mixture to 10 mL. All samples were shaken at 100 rpm in a water bath at 37°C for 2 h. UV absorbance was recorded at 282 nm, and each measurement was performed in triplicate. The apparent binding constant (K_app) of CGA with LF can be calculated through Equation (2) [67].

$$1/\left({\mathrm{A}}_{\mathrm{obs}}-{\mathrm{A}}_0\right)=1/\left({\mathrm{A}}_{\mathrm{c}}-{\mathrm{A}}_0\right)+1/\left({\mathrm{A}}_{\mathrm{c}}-{\mathrm{A}}_0\right)\times 1/{\mathrm{K}}_{\mathrm{app}}\left[\mathrm{CGA}\right]$$ (2)

where A_obs is the absorbance values of LF‐CGA at the different concentrations of CGA, A₀ is the absorbance value of LF and A_c is the absorbance value of LF‐CGA. α is the association degree between LF and CGA.

4.5.3. Intrinsic Fluorescence Spectra

The CGA solution (0.1 mM) with different volumes (0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mL) was mixed with 1 mL of LF solution (4 mg/mL). The distilled water was added to the final total volume of 10 mL. The samples were shaken at a speed of 100 rpm in a thermostatted shaker at different temperatures (298, 304, and 310 K) for 2 h. Fluorescence spectra were collected with an excitation wavelength of 293 nm, and emission signals were recorded over the range of 300–450 nm [67]. Generally, Equations (3) and (4) were used to evaluate the dynamic and static quenching process of intramolecular affinity. The intramolecular affinity of CGA and LF was evaluated by Equation (5) [67].

$${\mathrm{F}}_0/\mathrm{F}=1+{\mathrm{K}}_{\mathrm{q}}{\tau }_0\left[\mathrm{CGA}\right]=1+{\mathrm{K}}_{\mathrm{sv}}\left[\mathrm{CGA}\right]$$ (3)

where F₀ represents the fluorescence intensity of LF, F represents the fluorescence intensity of LF with CGA, K _q represents the molecular quenching rate constant, τ₀ represents the fluorescence lifetime of LF without quencher, which is about 10⁻⁸ s, K_sv is the Stern–Volmer quenching constant, and [CGA] represents the concentration of CGA.

$${\mathrm{F}}_0/\mathrm{\Delta }\mathrm{F}={\mathrm{F}}_0/\left({\mathrm{F}}_0-\mathrm{F}\right)=1/{\mathrm{f}}_{\mathrm{a}}+1/{\mathrm{f}}_{\mathrm{a}}{\mathrm{K}}_{\mathrm{a}}\times 1/\left[\mathrm{CGA}\right]$$ (4)

where F₀ is the fluorescence intensity of LF, F means the fluorescence intensity of LF with CGA, f_a is the fluorescence fraction, and K _a represents effective quenching rate constant.

The binding constant and the number of binding sites of CGA to LF were calculated through Equation (5).

$$\lg \left[({\mathrm{F}}_0-\mathrm{F})/\mathrm{F}\right]=\lg {\mathrm{K}}_{\mathrm{b}}+\mathrm{nlg}\left[\mathrm{CGA}\right]$$ (5)

where F₀ is the fluorescence intensity of LF, F is the fluorescence intensity of LF with CGA, K _b is the binding constant, and n is the number of binding sites.

4.5.4. Thermodynamic Analysis

Thermodynamic parameters involve changes in Gibbs free energy ($\mathrm{\Delta }G$), enthalpy ($\mathrm{\Delta }H$), and entropy ($\mathrm{\Delta }S$) [71]. Thermodynamic parameters were calculated using the Van't Hoff equation, shown as Equations (6) and (7) [71].

$$\ln {\mathrm{K}}_{\mathrm{a}}=-\mathrm{\Delta }H/\mathrm{RT}+\mathrm{\Delta }S/\mathrm{R}$$ (6)

$$\mathrm{\Delta }G=\mathrm{\Delta }H-\mathrm{T}\mathrm{\Delta }S$$ (7)

where K _a is effective quenching rate constant, R is the universal gas constant, T is the absolute temperature.

4.5.5. Molecular Docking

The crystal structure of LF was retrieved from the protein data bank (PDB) and pretreated with PyMOL 2.3.0 to remove bound water and native ligands. The molecular structure of CGA was obtained from the PubChem database, and energy minimization was conducted using ChemBio 3D Ultra 14.0. Molecular docking between LF and CGA was performed using AutoDock Vina 1.1.2 [26].

4.6. Fabrication of ELC/PE

ELC/PE was prepared following previous reported methods [72]. The LF‐CGA complex was dissolved in distilled water to achieve a final concentration of 1.5 wt% in the aqueous phase. The volume ratio of aqueous phase to EG was 15:1. The EG was added drop‐by‐drop and further stirred at a speed of 500 rpm for 0.5 h. The mixture was homogenized at a speed of 15,000 rpm for 2 min on a high‐speed homogenizer (IKA, Germany) to obtain ELC/PE.

4.7. Particle Size and Zeta Potential of ELC/PE

The particle size and Zeta potential were determined by DLS analysis (Shanghai Spectris Instruments System Co., Ltd., Nano ZS, China), as reported in previous studies [66].

4.8. EG Content and Emulsion Type of ELC/PE

HPLC instrument equipped with a Shim‐pack GISS C18 column (250 × 4.6 mm, 5 µm) at a column temperature of 25°C. The flow rate was 1.0 mL/min. The method used a binary gradient elution from 32% to 80% methanol in distilled water over 17 min (Table S1). The injection volume was 10 µL. The preparation of the standard and sample solutions was provided in Supporting Information.

4.9. Antioxidant Activity in Vitro

The scavenging rates of DPPH⁻, O₂ ⁻, ABTS⁺, and Fe³⁺ were measured using a spectrophotometer (Shanghai METASH Instruments Co., Ltd., UV‐5500, China). Seven experimental groups were set: control (CON), olive oil (OO), LF‐stabilized blank PE (LF/PE), LF‐CGA‐stabilized blank PE (LC/PE), EG, LF‐stabilized EG PE (ELF/PE), and LF‐CGA‐stabilized EG PE (ELC/PE). All the following methods and equations were followed the assay kits instructions.

4.9.1. DPPH⁻ Free Radical Scavenging Rate

Equal volumes (2 mL) of LF or LF‐CGA aqueous solution and 2 mL of DPPH⁻ solution (4 mg/mL) were mixed and incubated in the dark at room temperature for 30 min. The absorbance was measured at 517 nm by a UV spectrophotometer (Shanghai Metash Instruments Co., Ltd., UV‐5500, China). The scavenging rate of DPPH⁻ radicals was calculated using Equation (8).

$$\mathrm{The}\mathrm{scavenging}\mathrm{rate}\left(\%\right)=\left[1-\left({\mathrm{A}}_1-{\mathrm{A}}_2\right)/{\mathrm{A}}_0\right]\times 100$$ (8)

where A₁ is the absorbance measured at 517 nm wavelength, A₀ is the absorbance of the ethanol solution in the absence of DPPH⁻. A₂ is the absorbance of the sample solution with ethanol.

4.9.2. O₂ ⁻ Free Radical Scavenging Rate

0.25 mL of LF or LF‐CGA aqueous solution (4 mg/mL) was mixed with 4.5 mL of Tris‐HCl buffer (pH 8.2) and maintained at 25°C for 20 min. Pyrogallol solution was then added to the solution, which was terminated after 5 min by adding 1 mL of 8 mmol/L HCl. Subsequently, 1 mL of HCl (8 mmol/L) was added to determine the absorbance at 320 nm wavelength. The O₂ ⁻ radical scavenging rate was measured according to Equation (9).

$$\mathrm{The}\mathrm{scavenging}\mathrm{rate}\left(\%\right)=\left[1-\left({\mathrm{A}}_1-{\mathrm{A}}_2\right)/{\mathrm{A}}_0\right]\times 100$$ (9)

where A₀ is the absorbance of replacing the sample solution with distilled water, A₁ is the absorbance of the sample with pyrogallol solution, A₂ is the absorbance of replacing the pyrogallol solution with distilled water.

4.9.3. ABTS⁺ Free Radical Scavenging Rate

ABTS⁺ solution (7 mM) was mixed with K₂S₂O₈ solution (2.45 mM) and incubated in the dark for 12 h to prepare the ABTS radical working solution. LF and LF‐CGA aqueous solutions were added to the ABTS⁺ radical working solution. The mixture was reacted under light for 6 min. The absorbance was recorded at 734 nm. Results were expressed as Trolox equivalents (µg Trolox/mL sample).

4.9.4. Fe³⁺ Free Radical Scavenging Rate

0.4 mL of Trolox solution was mixed with 2.5 mL of phosphate buffer (pH 6.5) and 2.5 mL of K₃[Fe(CN)₆] solution. The mixture was incubated at 50°C for 20 min, followed by the addition of 2.5 mL of trichloroacetic acid. The sample was centrifuged at 3000 rpm for 10 min. Subsequently, 2.5 mL of the supernatant was mixed with 2.5 mL of distilled water and 0.5 mL of FeCl₃ solution at 25°C for 10 min. The absorbance of the resultant solution was determined at 700 nm wavelength. Results were expressed as Trolox equivalents (µg Trolox/mL sample).

4.10. Antibacterial Ability In Vitro

Antibacterial activity against C. albicans and S. aureus was evaluated using the quantitative suspension method. The experimental groups were consistent with those described in Section 4.9. Briefly, 5 mL of each sample was transferred to a sterile test tube and placed at 20°C for 5 min. Subsequently, 0.1 mL of bacterial suspension (5 × 10⁵ CFU/mL) was added and fully mixed. After incubation for 0.5 h, 1.0 mL of the mixture was spread onto agar plates and incubated at 37°C for 48 h. The bacterial inhibition rate was calculated through the plate count method according to Equation (10) [73].

$$\mathrm{Bacterial}\mathrm{inhibition}\mathrm{rate}\left(\%\right)=\left({\mathrm{A}}_0-{\mathrm{A}}_1\right)/{\mathrm{A}}_0\times 100$$ (10)

where A₀ is the number of viable bacteria (CFU/mL) in the positive control group, A₁ is the number of viable bacteria (CFU/mL) in the sample group.

4.11. Mouse Ear Edema Assay

The mouse ear edema model was established in KM mice to evaluate the anti‐inflammatory effect of the PE. The methods followed the previous study [74], with slight modifications. Thirty‐two male mice (∼22 g) were taken and housed in separate cages for 3 d (T = 22°C, RH = 50%–60%), with auto‐regulated day and night for 12 h each, and free access to food and water. The mice were randomly divided into eight groups (n = 4) as follows: normal control (NC), model control (MC), OO, LF/PE, LC/PE, EG, ELF/PE, ELC/PE, and positive control (PC). 0.04 mL of Xylene was applied to the right ear of each group to induce ear edema and maintained for 15 min. After 15 min, corresponding samples were administered to the right ear at 0, 1, and 2 h post modeling. The mice's right ear pieces (d = 9 mm) were obtained. The ear swelling and the inhibition rates were calculated using Equations (11) and (12) [74].

$$\mathrm{S}={\mathrm{M}}_1-{\mathrm{M}}_0$$ (11)

$$\mathrm{Inhibition}\mathrm{rate}\left(\%\right)=\left({\mathrm{S}}_0-{\mathrm{S}}_1\right)/{\mathrm{S}}_0\times 100$$ (12)

where M₀ represents the weight of the left ear, M₁ represents the weight of the right ear. S₀ represents the ear swelling weight in the control group. S₁ represents the ear swelling weight in the drug application groups.

4.12. Anti‐inflammatory Ability In Vivo

The activities of SOD, MPO, and NO in ear tissues were determined using assay kits, following the kits' instructions. All absorbance readings were acquired at 550 nm using a UV spectrophotometer. The activities of SOD, MPO, and NO were measured through Equations (13) to (15) mentioned on the instructions.

$$\mathrm{SOD}\mathrm{Viability}=({\mathrm{A}}_0-{\mathrm{A}}_1)/2{\mathrm{A}}_0\times {\mathrm{V}}_0/{\mathrm{V}}_1$$ (13)

$$\mathrm{MPO}\mathrm{Viability}=\left({\mathrm{A}}_1-{\mathrm{A}}_0\right)/11.3\times \mathrm{W}$$ (14)

$$\mathrm{NO}\mathrm{Viability}=\left({\mathrm{A}}_1-{\mathrm{A}}_0\right)/\left({\mathrm{A}}_2-{\mathrm{A}}_0\right)\times {\mathrm{C}}_0\times \mathrm{Sample}\mathrm{dilution}\mathrm{ratio}$$ (15)

where A₀ is the absorbance of the control group, A₁ is the absorbance of the samples, and A₂ is the absorbance of the standard group. V₀ is the volume of the reaction solution, and V₁ is the volume of the sample. C₀ is the concentration of the standard sample.

4.13. Statistical Analysis

All the data were analyzed with SPSS 22.0 software, and the results are expressed as the means ± standard deviations (SD). Furthermore, t‐tests and one‐way ANOVA were used for statistical analysis. *p < 0.05, **p < 0.01, indicate a significant difference. Graphs were prepared in Origin 2021.

Author Contributions

Xiyao Dong: writing – original draft, writing – review & editing, data curation, formal analysis. Tongyan Liu: conceptualization, methodology, data curation, formal analysis. Mengfan Li: writing – original draft, software. Xinying Wang: methodology, writing – review & editing. Qingxiang Guan: supervision, resources, project administration, funding acquisition .

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting Information

Supporting Information for this article is in the attached file.

Supporting information

Supporting File 1: cbdv71588‐sup‐0001‐SuppMat.pdf

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.