Inclusion complex of berberine hydrochloride with serine-β-cyclodextrin: construction, characterization, inclusion mechanisms, and bioactivity

Dong Ju Zhou† ^a, Wei Ming Liu†^a, Su Ping Dai^a, Shuai Qiang Jiang^a, Yin Wang^a, Jia Jia Yang^a, Ya Wei Chen^a, Jun Liang Chen^b, Hyun Jin Park^c and Hui Yun Zhou*^a
aSchool of Chemistry & Chemical Engineering, Henan University of Science and Technology, 263 Kaiyuan Avenue, Luoyang 471023, China. E-mail: hyzhou@haust.edu.cn; Tel: +86-0379-64231914
^bCollege of Food and Bioengineering, Henan University of Science and Technology, Luoyang 471023, China
^cGraduate school of Biotechnology, Korea University, 1,5-Ka Anam-Dong, Sungbuk-ku, Seoul 136-701, Korea

First published on 16th August 2024

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1. Introduction

Cyclodextrins (CDs), namely α (6)-, β (7)-, and γ (8)-CDs, are cyclic oligosaccharides formed by the linkage of glucose units through α-1, 4-glucosidic bonds with a hydrophobic inner cavity and hydrophilic outer surface.^1,2 They are widely applied in medicine, food, cosmetics, household products, textile, and agriculture.³ However, the volume of the α-CD inner cavity is inadequate, it is difficult for γ-CD to pass through biological membranes, and γ-CD is expensive.⁴ In contrast, β-CD is widely used because of its suitable cavity size (guest molecular weight between 200 and 800 g mol⁻¹) and low price.⁵ Hydrophobic guest molecules can be complexed with β-CD through non-covalent interactions to improve its stability, solubility, and bioavailability as well as to mask its unpleasant taste and smell.^6–8

However, natural β-CD has lower water solubility, higher hemolytic ability and renal toxicity.⁹ Therefore, it is necessary to modify β-CD to improve its properties for effective application. β-CD derivatives, including hydroxypropyl-β-CD, sulfobutylated-β-CD, randomly methylated-β-CD, and maltosyl-β-CD, have been developed.^10,11 Alexandre et al. prepared amiodarone hydrochloride and methyl-β-CD inclusion complexes through a spray-drying method to improve the solubility and dissolution rate of the drug.¹² Li et al. prepared polyphenols and sulfobutyl ether-β-CD inclusion complexes using a freeze-drying method to improve the stability and in vitro bioactivity of polyphenols.¹³

Amino acids are the building blocks of proteins and include alkaline amino groups and acidic carboxyl groups, which can be divided into non-polar and polar; polar amino acids can be further divided into neutral, acidic and basic. Amino acid modification can improve the properties of cyclodextrins.^14,15 We have previously studied inclusion complexes prepared from β-CD modified with basic amino acids (L-lysine). L-Serine (Ser) is a neutral non-essential amino acid and has a hydroxyl group (–OH) in the side chain, which is directly involved in cell homeostasis, proliferation and differentiation.¹⁶ To further expand the applicability of β-CD, we used a green and simple method to synthesize serine-modified β-CD derivatives.

Berberine hydrochloride (BBH) is a natural alkaloid (isoquinoline) with yellow needle-shaped crystals that can be extracted from the roots, rhizomes, and stem skins of Berberidaceae, goldenseal, Menispermaceae, and other plants (Coptidis rhizoma, Phellodendri chinensis cortex, and Berberidis radix).^17,18 BBH has been reported to have several bioactivities, including antibacterial, anti-inflammatory, antiviral, anti-tumor, and antioxidant activities, and is commonly used for treating diarrhea, dysentery, and various gastrointestinal infections.^19,20 However, BBH has poor water solubility, low oral bioavailability, and gastrointestinal malabsorption.²¹ To address these limitations of BBH, researchers have proposed approaches involving a variety of carriers, such as liposomes, micelles, solid dispersions, dendrimers, nanocarriers, and inclusion complexes.^20,22 Cyclodextrin inclusion complexes have a broad application prospect in improving the properties of hydrophobic drugs. Xiao et al. prepared BBH/β-CD inclusion complexes and characterized them using different techniques.²³ Popiołek et al. synthesized a cationic derivative of γ-cyclodextrin modified with propylenediamine and formed an inclusion complex for berberine.²⁴ Zhang et al. investigated the role of β-CD in improving the intestinal absorption of berberine hydrochloride, a P-glycoprotein substrate.²⁵ Tong et al. prepared microspheres of BBH and trimethoprim by cross-linking chitosan with sulfobutylether-β-CD and characterized them.²⁶ Jia et al. reported the fluorescence enhancement of BBH when complexed with β-CD.²⁷ Ma et al. prepared the inclusion complex of hydroxypropyl-β-CD in order to reduce the bitterness of BBH.²⁸ However, there have been no reports on the preparation and characteristics of inclusion complexes of BBH and serine-modified β-CD.

In this paper, a β-CD derivative modified with Ser was synthesized, and the BBH/Ser-β-CD IC was prepared by a freeze-drying method. The inclusion complexes were characterized, and the release behavior of BBH from the IC was studied. The host–guest combination model and interaction mechanism were explored by molecular docking. In addition, the biocompatibility and antibacterial properties of the BBH/Ser-β-CD IC against E. coli and S. aureus were evaluated.

2. Materials and methods

2.1. Materials

β-Cyclodextrin (β-CD, 99.0%) and L-serine (Ser, 99.0%) were provided by Yuanye Bio-Technology Co., Ltd. p-Toluene sulfonyl chloride (TsCl, 99.0%) and berberine hydrochloride (BBH, 98.0%) were provided by Aladdin (Shanghai, China). Triethylamine (TEA, 99.5%) and ethyl alcohol (EtOH, 99.7%) were purchased from Damao chemical reagent Co., Ltd. (Tianjin, China). S. aureus (ATCC6538) and E. coli (ATCC8739) strains were supplied by the School of Chemistry & Chemical Engineering.

2.2. Synthesis and characterization of mono-(6-L-serine-6-deoxy)-β-CD

Mono-(6-L-serine-6-deoxy)-β-CD (Ser-β-CD) was synthesized by the reaction of O-p-toluenesulfonyl-β-cyclodextrin (TsCD) and Ser. The methods were according to our previously reported.¹⁴ β-CD and TsCl were reacted in an alkaline solution at 0–5 °C, and the pH of the filtrate was adjusted to 6–7; the solid product was precipitated by refrigeration and filtered to obtain the crude product. The solid obtained by recrystallization was dried in a vacuum to obtain TsCD.

TsCD and Ser were dissolved in a 40% TEA solution and reacted at 85 °C under an N₂ atmosphere for 18 h. The product was centrifuged and the filtrate is rotated to obtain an oily liquid, which was poured into absolute ethyl alcohol. The filtered precipitates were separated and purified using a chromatographic column. Ser-β-CD was obtained by drying the target product under a vacuum, and the chemical equations of the synthesis process are depicted in Fig. 1A.

Ser-β-CD was characterized by FTIR and nuclear magnetic resonance (NMR). The FT-IR spectra were recorded at room temperature in the range of 4000 to 400 cm⁻¹ using an FTIR spectrometer (IR Tracer-100, Japan) by the KBr method.²⁹ The spectra were obtained at a resolution of 2 cm⁻¹ and as the average of 64 scans. The ¹H NMR spectra were recorded at 298 K on a Bruker AVANCE III (400 MHz, Germany) instrument using D₂O as the solvent.

2.3. Phase solubility study and water solubility test of Ser-β-CD

The phase solubility study was carried out according to the method reported by Higuchi and Connors.³⁰ Briefly, excessive BBH was mixed with a series of aqueous solutions containing increasing amounts of Ser-β-CD (0–20 mM) and dispersed using ultrasound for 5 min. Then, the suspensions were oscillated in an oscillating incubator (ZHSY-50N, China) at 25 °C for 48 h. After equilibrium was achieved, the samples were filtered through a 0.22 μm hydrophilic membrane filter. The BBH concentration was determined using an ultraviolet-visible spectrophotometer (UV-6100 Double Beam, Mapada, China). The phase solubility graph was drawn to understand the relationship between the concentration of drugs (mM) and the concentration of Ser-β-CD (mM). The stability constant K_S (L mol⁻¹) was computed using the slope and intercept using eqn (1):

	(1)

where the intercept is the intrinsic solubility of the drug.

The solubility of Ser-β-CD in water was investigated according to the solubility determination method in the Chinese Pharmacopoeia 2020. β-CD (80.0 mg) and Ser-β-CD (0.4 g, 0.8 g, and 1.2 g) were added to a small vial containing 2 mL distilled water, respectively. The resultant mixtures were stirred at 25 °C for 1 h and oscillated for 30 s every 5 min. The dissolution was observed within 30 min. Ser-β-CD was considered completely dissolved when no Ser-β-CD was observed in the solution.

2.4. Preparation of the inclusion complex

BBH/Ser-β-CD inclusion complexes (IC) were prepared by a freeze-drying method.³¹ BBH was dissolved in hot water (70 °C) and then added in drops to the Ser-β-CD solution. After magnetic stirring at a controlled temperature for a certain time, the suspension was filtered through a 0.22 μm hydrophilic membrane. The filtrate was freeze-dried in a vacuum freeze dryer (SCIENTZ-12 N/A, China) to obtain the BBH/Ser-β-CD IC. The process of preparation and performance testing of BBH/Ser-β-CD IC are depicted in Fig. 1B.

In addition, BBH and Ser-β-CD at a molar ratio of 1:1 were mixed in a vortex mixer for 10 min to obtain the physical mixture (PM).

2.5. Characterization of the inclusion complex

The surface morphologies of Ser-β-CD and the inclusion complexes were observed by scanning electron microscopy (SEM, HITACHI Flex SEM-1000, Japan). The samples were secured to the sample table using a conductive adhesive and coated with gold. The test was performed under high-vacuum conditions at an acceleration voltage of 10 kV.¹⁴ BBH, the physical mixture, and the inclusion complex samples were characterized by FTIR spectroscopy in the wavenumber range of 4000–400 cm⁻¹.³²

2.6. Molecular docking

To investigate the molecular interactions between Ser-β-CD and BBH, molecular docking was performed with AutoDock4.2.6 (Scripps Research Institute, La Jolla, CA, USA).⁵ The molecular structure of β-CD and BBH were extracted from the protein data bank (PDB:4YEF) and the PubChem database (CID:12456), respectively.³³ The structure of Ser-β-CD was established based on β-CD using the Gauss View software (Gaussian, Inc., Wallingford CT, USA). The β-CD molecule was first fixed, and according to the mechanism of the nucleophilic substitution reaction, the hydroxyl group at position 6 in a glucose unit was replaced by a serine outside the molecule.³⁴ The geometric configuration optimization and frequency calculations of Ser-β-CD and BBH were carried out at the level of DFT/B3LYP/6-31G* basis set by using the Gaussian 16 package.³⁵

The molecular docking analysis was performed using AutoDock4.2.6 with the optimized 3D structure of Ser-β-CD as the receptor and the optimized 3D structure of BBH as the ligand, and the configuration of minimum binding energy was obtained. The ligand and receptor files were processed using AutoDock Tools; the van der Waals and electrostatic energy grid maps were created by AutoGrid to cover the entire receptor, and the Lamarck genetic algorithm (LGA) was used to search for the binding model with the lowest free energy.^31,36,37 Open Babel GUI was used to convert the correct structural format.³⁸

2.7. In vitro release studies

The release of the drug from the prepared inclusion complexes was studied using the method reported by Ren et al.¹⁵ The dialysis bag containing the inclusion complexes (10 mg) was placed in a conical bottle containing 40 mL PBS (0.2 mol L⁻¹, pH 6.8) and oscillated at 100 rpm and a temperature of 37 °C; a 5-mL aliquot was removed at predetermined intervals, and fresh release medium was replenished to maintain a constant volume. The absorbance of the samples was measured at 357 nm by UV-vis spectrometry, and the BBH concentration was assayed. The cumulative release efficiency (Er%) was calculated using eqn (2):

	(2)

where C_i and C_n are the sample concentration in the aliquot, V_e is the volume of mediator taken, V₀ is the total volume of PBS, m₀ is the total amount of drug in the ICs, and n is the number of samples taken.

2.8. Hemolysis assays

The hemolysis test was used to evaluate the blood compatibility of Ser-β-CD and BBH/Ser-β-CD IC.^39,40 The animals and protocols used in this study were approved by the Experimental Animal Ethics Committee of Henan University of Science and Technology (approval no. 20231119). Fresh blood (5 mL) was collected from mice (5 Kunming mice, 18–25 g) and placed in heparinized tubes. The obtained blood samples were centrifuged (1500 rpm, 10 min) to remove plasma, white cells, and platelets. The red blood cells were diluted with physiological saline (1:1, v/v) and centrifuged under the same conditions; the process was repeated three times. The precipitate was formulated to prepare a 2% red blood cell (RBC) suspension.⁴¹ Different concentrations of Ser-β-CD or BBH/Ser-β-CD IC solution (50, 100, 250, 500, and 1000 μg mL⁻¹) were prepared using physiological saline, respectively. The RBC suspension (400 μL) was mixed with Ser-β-CD (400 μL) or BBH/Ser-β-CD IC (400 μL). The physiological saline (400 μL) served as the negative control, and a 0.1% Triton X-100 (400 μL) solution was used as the positive control. The samples and controls were incubated at 37 °C for 3 h and then centrifuged (1500 rpm, 10 min). The supernatants were added to a 96-well plate, and the absorbance was measured at 570 nm using a microplate reader. Each sample was measured in triplicate, and the hemolysis rate was calculated using eqn (3).⁴²

	(3)

2.9. Antibacterial activities evaluation

The optical density method was used to evaluate the antibacterial effects of the BBH/Ser-β-CD inclusion complexes in Luria–Bertani (LB) liquid medium against E. coli (model Gram-negative bacterium) and S. aureus (model Gram-positive bacterium).^41,43,44 Different concentrations of BBH/Ser-β-CD IC aqueous solutions were added to 10 mL of LB medium containing E. coli suspension (0.2 mL, 10⁵–10⁶ CFU mL⁻¹) or S. aureus suspension (2 mL, 10⁵–10⁶ CFU mL⁻¹), respectively. The suspensions were cultured in an oscillating incubator (HZQ-F160, China) at 180 rpm at 37 °C for 24 h. At a predetermined time, the OD values of the suspensions at 600 nm were determined by UV-vis spectrometry.⁴⁵ The penicillin–streptomycin solution was used as the reference to assess and validate the effectiveness of the inclusion complex as an antibacterial agent.⁴⁶ The penicillin–streptomycin solution (1000 μL) was added to LB medium (10 mL) containing E. coli suspension (0.2 mL, 10⁵–10⁶ CFU mL⁻¹) or S. aureus suspension (2 mL, 10⁵–10⁶ CFU mL⁻¹), which acted as the positive control group.

2.10. Statistical analysis

All data are expressed as mean ± standard deviation (S.D.). Each experiment was carried out in triplicate. Statistical analysis was conducted using one-way analysis of variance (ANOVA).

3. Results and discussion

3.1. Characterization of Ser-β-CD

The FTIR spectra of Ser-β-CD are shown in Fig. 2A. The FT-IR spectrum of β-CD showed characteristic bands belonging to saccharides at 945 cm⁻¹ (α-pyran ring), 1155 cm⁻¹ (C–O–C symmetrical stretching), 1080 cm⁻¹ and 1030 cm⁻¹ (C–O and C–O–C stretching vibrations, respectively).^47,48 The FTIR spectrum of Ser had peaks at 3500–3400 cm⁻¹ (C–O and –OH stretching vibrations), 1601 cm⁻¹ (CO stretching vibration), 1498 cm⁻¹ (–NH₂ bending vibration), 1410 cm⁻¹ (COO⁻ stretching vibration), and 1343 cm⁻¹ (bending vibration). In the spectrogram of Ser-β-CD, the peak of the symmetrical stretching vibration generated by COO⁻ had blue-shifted to 1412 cm⁻¹, while the peak of –CH₂ vibration had blue-shifted to 1345 cm⁻¹ and the peak deformation was wide.

The ¹H NMR spectrum of Ser-β-CD is shown in Fig. 2B. The chemical shift of some acidic hydrogen nuclei, such as active hydrogen (–OH, –COOH, –NH–, and –SH), which are connected to electronegative atoms, such as O, N, and S, was greatly affected by the solvent and temperature, and they disappeared with the addition of D₂O. The multiple peaks at 5.16–4.96 ppm were produced by hydrogen at position 1 of the pyran ring in the CD parent body. The multiple peaks at 4.54–4.44 ppm and 4.22–4.14 ppm were caused by the increase in the chemical shift of –CH₂, which is connected to Ser on the CD, under the influence of Ser. The peak of hydrogen on the chiral carbon in Ser ranged from 4.02 to 3.71 ppm. The multiple peaks at 4.02–3.71 ppm and 3.64–3.49 ppm were generated by the remaining hydrogen atoms on C2, C3, C4, C5, and C6 of the CD parent body.

3.2. Phase solubility and water solubility of Ser-β-CD

According to Higuchi and Connors, the solubilizing capacity, apparent stability constant, and stoichiometric ratio of Ser-β-CD to BBH were investigated by phase solubility.⁴⁹ The phase solubility diagram of aqueous BBH solutions at different Ser-β-CD concentrations at 25 °C is shown in Fig. 3A. The solubility of BBH increased linearly with the increase in Ser-β-CD concentration, exhibiting a solubility curve of A_L type.^50,51 Moreover, the slope of the curve was less than one, confirming that the drug was complexed with CD at a 1:1 stoichiometric ratio.⁵² The stability constant (K_S) calculated using eqn (1) was 173.9 M⁻¹, which reflects that the strength of host–guest bonding is suitable for the preparation of stable inclusion complexes. As per previous reports, when the value of K_S is between 50 and 2000 M⁻¹, stable clathrates are formed.⁵³

The water solubility of β-CD and Ser-β-CD was evaluated in water. As shown in Fig. 3B, β-CD (40 mg mL⁻¹) showed significant turbidity in water, while Ser-β-CD (200, 400, and 600 mg mL⁻¹) formed clear solutions under the same condition. Ser-β-CD showed excellent water solubility even at a high concentration of 600 mg mL⁻¹. The solubility of β-CD in water was only 18.5 mg mL⁻¹, and the intrinsic solubility of Ser in water was 250 mg mL⁻¹. Ser-β-CD significantly improved the solubility of both β-CD and Ser. The reason might be that the Ser in β-CD destroys the hydrogen bonding of β-CD, thereby changing its original rigid structure and resulting in improved water solubility.

3.3. SEM analysis

The surface morphology of BBH, Ser-β-CD, BBH/Ser-β-CD PM, and BBH/Ser-β-CD IC was observed by SEM (Fig. 4). The BBH powder (A) was irregularly square in shape with an obvious crystal structure.²² Ser-β-CD (B) presented an amorphous morphology with irregular parallelogram shapes of different sizes and relatively dense structures. The images of PM (C) showed that BBH and Ser-β-CD retained their original morphology and size. The images of BBH/Ser-β-CD IC (D) revealed irregular sheets, different from those of Ser-β-CD and PM. Hasanvand et al. reported that the surface morphology changed after vanillin and β-CD formed the inclusion complex, consistent with our observation.⁵⁴ The inclusion of the drug in CD changed the original conformation, and differences in the microstructure confirmed the formation of the inclusion complexes.⁵⁵ Thus, BBH/Ser-β-CD IC was successfully prepared.

3.4. FT-IR spectra analysis

The interaction between the host and guest molecules in the solid-state inclusion complex was analyzed by the FT-IR technique.⁵⁶ When the guest molecules enter the cyclodextrin cavity, the shape, intensity, and position of the peaks are known to change (alter or vanish).⁵⁷ The recorded infrared spectra of BBH, Ser-β-CD, BBH/Ser-β-CD PM, and BBH/Ser-β-CD IC are shown in Fig. 5. The characteristic infrared peaks of BBH were found at 1615 cm⁻¹ (CN⁺ stretching vibration), 1178 cm⁻¹ and 1132 cm⁻¹ (C–O stretching vibrations).⁴³ The characteristic absorption peaks of BBH and Ser-β-CD were found in the infrared spectrum of the physical mixture, indicating that they were simply stacked. In the infrared spectrum of the IC, the peak caused by the C–O stretching vibration had disappeared, and the absorption peak of the quaternary ammonium ion CN⁺ was redshifted to 1588 cm⁻¹ under the influence of the electrostatic effect. Giselle et al. reported that the intensity and shape of the nifurtimox (NFX) peaks were changed after NFX formed clathrates with β-CD and sulfobutylether-β-CD.⁴⁸ Therefore, these findings confirmed that the inclusion complex was successfully prepared.

3.5. Molecular docking studies

Molecular docking was used to study the interaction between the host and guest molecules, effectively predict the bound conformations and binding affinity, and evaluate the binding potential of BBH in the Ser-β-CD cavity. The results of molecular docking are shown in Fig. 6A. The 9,10-dimethoxy-benzo group of BBH first enters the wide rim of Ser-β-CD and points to the narrow rim of the cavities of Ser-β-CD (the 1,3-benzodioxole group points to the wide rim of the cavities of Ser-β-CD). BBH completely enters the interior of the Ser-β-CD cavity with a hydrogen bond distance of 2.0 Å.

The minimum binding affinity of BBH with Ser-β-CD inclusion complexes was calculated as −7.87 kcal mol⁻¹, as shown in Table 1. The binding energy is the sum of intermolecular energy, internal energy and torsional energy minus the unbound energy.³⁸ The negative binding affinity value indicates that BBH binds well to Ser-β-CD, and the inclusion complex has higher stability.^6,36 The two-dimensional images of the BBH-CD inclusion complexes are shown in Fig. 6B, where the dashed lines represent hydrogen bonds, and the circular arcs represent hydrophobic contacts.⁵⁸

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3.6. In vitro release studies

The release profiles of BBH, BBH/Ser-β-CD PM, and BBH/Ser-β-CD IC are shown in Fig. 7. The cumulative release amount of BBH was 92% within 24 h, and the levels reached equilibrium at 3 h. The release behavior of PM was similar to BBH, and equilibrium was reached at 3 h. The equilibrium time of BBH/Ser-β-CD IC was 6 h, and the cumulative release was 77%. The results show that compared with free BBH and PM, the prepared ICs have slower release characteristics. Overall, the inclusion complexes prepared with BBH and Ser-β-CD may be a potential sustained-release system.

3.7. Hemolysis assays

The biocompatibility of Ser-β-CD and BBH/Ser-β-CD IC was evaluated by the hemolysis assay. As shown in Fig. 8, hemolysis occurred in the positive control group, while the solution remained clarified in the negative control group and the experimental group, with red blood cells accumulating at the bottom. Hemoglobin release is one of the key parameters used to evaluate the integrity of erythrocyte membranes.⁵⁹ A high hemolysis rate (5%) is a sign of hemolysis.⁴¹ The hemolysis rates of Ser-β-CD and BBH/Ser-β-CD IC were lower than 5%. When the concentration of Ser-β-CD was 1000 μg mL⁻¹, the hemolysis rate was lower than 3%. It has been reported that β-CD at a concentration of 1000 μg mL⁻¹ causes hemolysis up to a rate of 18%.⁶⁰ The results show that Ser-β-CD and BBH/Ser-β-CD IC have good blood compatibility, and the biocompatibility of β-CD can be improved by modifying it with Ser.

3.8. Antibacterial activity evaluation

The growth inhibition effect of different concentrations of BBH/Ser-β-CD IC on E. coli and S. aureus was investigated for 24 h, with bacteria grown in normal conditions as the control. The growth curves of the bacteria obtained by adding different concentrations of BBH/Ser-β-CD IC are shown in Fig. 9. The bacterial growth curves in the presence of Ser-β-CD was similar to that of the control group, indicating that Ser-β-CD had little inhibitory effect on the growth of E. coli and S. aureus. The PM had a certain inhibitory effect on the growth of E. coli and S. aureus. The inhibitory effect of BBH/Ser-β-CD IC on E. coli enhanced with the increase in IC concentration. When the concentration reached 5000 μg mL⁻¹, the growth of E. coli was completely inhibited within 4 h. A 10 μg mL⁻¹ BBH/Ser-β-CD IC solution could effectively inhibit S. aureus within 24 h. The bacteriostatic effect of BBH/Ser-β-CD IC on E. coli was weaker than that of the positive control up to 10 μg mL⁻¹. On the other hand, the BBH/Ser-β-CD IC solution could effectively inhibit S. aureus within 24 h when the concentration was higher than 10 μg mL⁻¹, similar to the positive control.

The BBH/Ser-β-CD IC showed a dose-dependent inhibitory effect on the growth of both E. coli and S. aureus. Its bacteriostatic effect on S. aureus is better probably because S. aureus has better sensitivity to the drug BBH.

In addition, the bacteriostatic effect of Ser-β-CD IC was better than that of Ser-β-CD or the physical mixture of BBH and Ser-β-CD. The results showed that the bacteriostatic effect might be dependent on the amount and state of the added BBH. The binding energy of the BBH and Ser-β-CD IC (−7.87 kcal mol⁻¹) indicated that BBH was well-included in Ser-β-CD, completely entering the interior of the Ser-β-CD cavity through hydrogen bonding, and the inclusion complex exhibited higher stability. Furthermore, Ser-β-CD showed excellent water solubility even at a concentration of up to 600 mg mL⁻¹, and the solubility of BBH increased linearly with an increase in Ser-β-CD concentration. The results of phase solubility studies showed that the stability constant of the BBH/Ser-β-CD IC was 173.9 M⁻¹, which is between 50 and 2000 M⁻¹, and stable inclusion complexes could be formed. Therefore, Ser-β-CD could form stable clathrates with BBH, and the dissolved amount of BBH could be increased by increasing the concentration of the Ser-β-CD solution. The results of the in vitro release study showed that the prepared BBH/Ser-β-CD IC was more efficient in sustained drug release compared with free BBH and PM due to the stability of the inclusion complex of BBH and Ser-β-CD. Therefore, the bacteriostatic effect of BBH/Ser-β-CD IC is sustainable because of the sustained release of the inclusion complex of BBH with Ser-β-CD. In conclusion, BBH/Ser-β-CD IC has prospective applications as an antibacterial agent.

4. Conclusions

Serine-modified β-CD derivatives were successfully synthesized and characterized by FT-IR and NMR. BBH/Ser-β-CD IC was prepared by the freeze-drying method. Phase solubility studies showed that BBH and Ser-β-CD formed stable inclusion complexes in a stoichiometric ratio of 1:1. According to SEM characterization, IC had an irregular sheet-like structure, different from those of Ser-β-CD and PM. The characteristic peak of BBH in the FT-IR spectrum of IC showed changes, which confirmed the successful preparation of BBH/Ser-β-CD IC. The results of molecular docking analysis showed that BBH was completely contained in the Ser-β-CD cavity, and the binding energy was −7.87 kcal mol⁻¹. The release rate of BBH from BBH/Ser-β-CD IC was slower than that of the free drug or from the PM, which indicates that BBH/Ser-β-CD IC might be a potential sustained release system. No hemolysis occurred in the presence of Ser-β-CD and BBH/Ser-β-CD IC, and the hemolysis rate was lower than 5%, indicating that the synthesized Ser-β-CD and IC had good biocompatibility. The BBH/Ser-β-CD IC exhibited antimicrobial activity against E. coli and S. aureus, and the antibacterial effect against S. aureus was better than that against E. coli. In conclusion, the BBH/Ser-β-CD IC has good biocompatibility and antibacterial properties, which enable its potential application in biomedicine and the food industry.

Author contributions

Dong Ju Zhou and Wei Ming Liu participated in the topic selection design, finished the experiment, and wrote the manuscript. Su Ping Dai, Shuai Qiang Jiang, Yin Wang, and Jia Jia Yang, completed the concrete analysis of the data and carried out the experimental verification. Ya Wei Chen and Jun Liang Chen provided research equipment and discussed the results. Hyun Jin Park reviewed and revised the manuscript. All authors examined and accredited the ultimate version of the manuscript. Hui Yun Zhou identified the research topic, designed the study and revised the manuscript.

Data availability

The data are available from the corresponding author on reasonable request.

Conflicts of interest

The authors report no conflicts of interest.

Acknowledgements

The authors are indebted to the financial assistance from the Natural Science Foundation of Henan Province (No. 182300410213).

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Footnote

† Dong Ju Zhou and Wei Ming Liu contributed equally to this work.

This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2024