Improvement of water solubility and antibacterial activity of Calophyllum inophyllum resin and calophylloidic acid A via inclusion complexation with cyclodextrins

Abstract

Indonesian Calophyllum inophyllum resin and its main component, calophylloidic acid A (1), have potent antibacterial activity against foodborne pathogens such as Staphylococcus aureus and Escherichia coli. However, their low water solubility makes their practical use difficult. Here, we investigated the formation of inclusion complexes of C. inophyllum resin and 1 with cyclodextrins (CDs). The inclusion complexes of C. inophyllum resin or 1 with CDs exhibited enhanced water solubility and antibacterial activity against S. aureus and E. coli compared to those of uncomplexed C. inophyllum resin or 1. Among the analyzed samples, α-CD inclusion complexes exhibited the strongest antibacterial activity. Furthermore, 1 and α-CD formed inclusion complexes at a molar ratio of 1:1. The improved water-solubilities and good antibacterial activity of the inclusion complexes will allow for their future application against foodborne pathogens.

Introduction

Food safety is an increasing concern because it has a major impact on global health. The World Health Organization estimates that approximately 10 % of the global population falls ill owing to the consumption of contaminated food, resulting in more than 420 000 deaths annually ⁱ⁾ (Wang et al., 2023). Pathogenic bacteria are major contributors to foodborne illnesses, and contamination by foodborne pathogens, such as Staphylococcus aureus and Escherichia coli can occur at various stages, including during food distribution, storage, retail, preparation, and serving (Callejas et al., 2023; Ouyang et al., 2023; Su et al., 2023). The inappropriate storage and cleaning of reusable plastic containers lead to cross-contamination via food and human hands, increasing the risk of foodborne illness (Kim et al., 2023). These factors must be considered to prevent infections caused by foodborne pathogens, particularly by ensuring hand hygiene and container washing.

Calophyllum inophyllum  is distributed in tropical regions worldwide, including Indonesia (Emilda, 2019; Govindasamy et al., 2022). Various parts of the Calophyllum species have been used in traditional medicines against diseases, such as skin infections and ulcers. In particular, the seed oil known as tamanu oil, is widely used for the treatment of skin diseases (Cottiglia et al., 2004; Gupta and Gupta, 2020). In addition, C. inophyllum shows potential for application as a biofuel and soil restoration; therefore, the future cultivation of C. inophyllum is expected to increase (Leksono et al., 2008). However, C. inophyllum resin is not currently being utilized commercially.

We have previously isolated calophylloidic acid A (1) was isolated from Indonesian C. inophyllum resin, as a major compound of the plant (Fig. 1). C. inophyllum resin and 1 possessed more potent antibacterial activity against the foodborne pathogens S. aureus and E. coli than conventional antibiotics, such as kanamycin and streptomycin (Mizuno et al., 2022). These results suggest that the potential application of C. inophyllum resin as daily products, such as soap and detergent with antibacterial activity. However, C. inophyllum resin is sparingly soluble in water, which limits its practical application in antibacterial products. Therefore, overcoming the issue of the low water solubility of the resin can lead to improvements in its antibacterial activity and utilization.

Fig. 1.

Structure of 1.

Cyclodextrins (CDs) are cyclic oligosaccharides consisting of multiple glucose linkages. There are three representative types of CDs, namely, α-, β-, and γ-CDs. CDs incorporate hydrophobic guest molecules into their cavities, thereby improving the solubility and bioactivity of the guest molecules (Cid-Samamed et al., 2022). CDs are widely used in pharmaceuticals, food, and cosmetics. For example, the water solubility, thermal stability, and herbicidal activity of cyanazine can be improved by the formation of inclusion complexes with β-CD (Gao et al., 2020), and the antibacterial activity of hinokitiol can be improved by forming inclusion complexes with α-, β-, and γ-CDs (Inoue et al., 2020). Therefore, CD inclusion complexes are also expected to improve the water solubility and antibacterial activity of C. inophyllum resin.

In this study, we prepared C. inophyllum resin and its CD inclusion complexes (C. inophyllum resin/CDs) using the co-precipitation method (Jiang et al., 2019). We confirmed the effectiveness of CDs on improving the water solubility and antibacterial activity of the resin against S. aureus and E. coli. In addition, we prepared 1 and its CD inclusion complexes (1/CDs) in a similar manner and evaluated their water solubility and antibacterial activity. Furthermore, the mechanism of the interaction between 1 and α-CD was investigated using spectroscopic analysis.

Materials and Methods

General experimental procedures  The 1D NMR spectra were obtained using a Bruker BioSpin AVANCE III (400 MHz) spectrometer (Bruker BioSpin, Rheinstetten, Germany), and chemical shifts were recorded in ppm. The NMR spectra were calibrated using the residual solvent peaks (TMS in DMSO: ¹H NMR 0 ppm). For analytical HPLC, a PU-2080 Intelligent HPLC pump, LG-2080-02 ternary gradient unit, UV-1570 Intelligent UV/VIS detector, and DG-980-50 degasser (all from Jasco, Tokyo, Japan) were used. The data were analyzed using the ChromatoPRO software (Lab Lab Co. Ltd., Tokyo, Japan). UV spectra were obtained using the UV spectrometer V-560 UV/VIS spectrophotometer (Jasco).

Materials  The ethanol (EtOH) extracts of Indonesian C. inophyllum resin and 1 obtained in our previous study (Mizuno et al., 2022) were used. The α-, β-, and γ-CDs were purchased from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan). All other reagents were of analytical grade.

Preparation of CD inclusion complexes via co-precipitation  CD inclusion complexes were prepared based on a previously reported co-precipitation method with minor modifications (Jiang et al., 2019). Each CD was dissolved in 50 % EtOH and stirred at 50 °C. The C. inophyllum resin extract or 1 were dissolved in EtOH and added dropwise to each stirred CD solution. The resin extract (assumed molecular weight 526.7) or 1 were weighed assuming that they form inclusion complexes with CDs at a molar ratio of 1:1. The mixture was stirred at 26 °C for 6 h and left overnight at 4 °C. The following procedures were different from C. inophyllum resin and 1. C. inophyllum resin/CDs was collected as powder by suction filtration. Then, the powder of C. inophyllum resin/CDs were washed with diethyl ether to remove exterior uncomplexed C. inophyllum resin extracts. After that, the powder of C. inophyllum resin/CDs were dried by vacuum pump. The inclusion rates of C. inophyllum resin/CDs were calculated gravimetrically, because Calophyllum resin are crude complexes. Each inclusion rate was 54.5 (α-CD), 30.3 (β-CD), and 85.6 % (γ-CD). On the other hand, for 1/CDs, we dried it without washing with diethyl ether (Zhou et al., 2021). As for 1/CDs, 1 adhered to CDs surface could not be washed with diethyl ether due to little sample amount of 1. Thus, the inclusion rates of 1/CDs could not be determined. We estimated that the inclusion rates of 1/CDs are 100 °%, and conducted all experiments for 1/CDs with the hypothesis.

Determination of minimal inhibitory concentration (MIC)  The MIC was determined as previously described (Mizuno et al., 2022). Briefly, each inclusion complex was prepared at concentrations of 5–2560 µg/mL in water, diluted 10-fold with each bacterial solution, and incubated at 37 °C for 24 h. The MIC was defined as the lowest concentration at which no turbidity was observed.

Identifying the presence of 1 in the C. inophyllum resin/CDs  C. inophyllum resin/CDs were sonicated with ethyl acetate to break the complexes and filtered using a 0.45µm membrane filter (Toyo Roshi Kaisha, Ltd., Tokyo, Japan). The ethyl acetate was evaporated, and the residue was dissolved in methanol and analyzed using HPLC. The HPLC conditions were as follows: Daicel CHIRALPAK IG column (10 × 250 mm, 5 µm); mobile phase water (A) and acetonitrile (B) containing 0.1 % formic acid; flow rate 1.0 mL/min; linear gradient, 10–100 % (B) in 45 min and held at 100 % for 15 min.

Phase solubility study  The phase solubility was determined using the Higuchi and Connors method (Higuchi and Connors, 1965). Various concentrations of aqueous α-CD solutions (0, 1, 2, 3, 4, 5, 6, 8, and 10 mM) were prepared, and then, an excess of 1 (approximately 20 mM) was added to each α-CD solution. Each sample was shaken at 26 °C for 68 h and filtered using a 0.45 αm membrane filter (Tokyo Roshi Kaisha, Ltd.). The concentration of 1 was calculated from the absorbance at 324 nm. From the obtained concentrations, graphs were prepared with the concentrations of 1 and α-CD on the vertical and horizontal axes, respectively.

Results

Determination of MICs of C. inophyllum resin/CDs  The MICs of C. inophyllum resin alone and C. inophyllum resin/CDs are listed in Table 1. Based on these results, the corrected MICs of C. inophyllum resin in the C. inophyllum resin/CDs considering the inclusion rates of each inclusion complex were calculated (Table 2). The CDs alone did not exhibit the antibacterial activity against S. aureus and E. coli confirmed by the paper disc method. The CD inclusion complexes exhibit a potent antibacterial activity against S. aureus and E. coli at lower amounts of C. inophyllum resin than that of C. inophyllum resin alone (Tables 1 and 2) confirming that the antibacterial activity of the C. inophyllum resin is improved by the formation of inclusion complexes with CDs. In particular, the C. inophyllum resin/α-CD exhibited the most notable improvement in the antibacterial activity.

Table 1 MICs (μg/mL) of the EtOH extracts of Calophyllum inophyllum resin and C. inophyllum resin/CDs inclusion complex against Staphylococcus aureus and Escherichia coli.

	S. aureus	E. coli
C. inophyllum resin	64	16
C. inophyllum resin/α-CD inclusion complex	64	32
C. inophyllum resin/β-CD inclusion complex	128	64
C. inophyllum resin/γ-CD inclusion complex	>256	128

Table 2 The calculated MICs of Calophyllum inophyllum resin (μg/mL) in C. inophyllum resin/CDs inclusion complex.

	S. aureus	E. coli
C. inophyllum resin/α-CD inclusion complex	12	6
C. inophyllum resin/β-CD inclusion complex	12	6
C. inophyllum resin/γ-CD inclusion complex	>63	32

Identifying the presence of 1 in the C. inophyllum resin/CDs  We broke C. inophyllum resin/CDs complexes with ethyl acetate, and determined the content of 1 in C. inophyllum resin/CDs by HPLC analysis. A peak corresponding to 1 is observed in the chromatograms of all C. inophyllum resin/CDs at a retention time of 37 min (Fig. 2). The C. inophyllum resin/α-CD chromatogram exhibits the most intense 1 peak.

Fig. 2.

HPLC chromatograms of Calophyllum inophyllum/cyclodextrin inclusion complex: C. inophyllum resin (a), C. inophyllum resin/α-CD inclusion complex (b), C. inophyllum resin/β-CD inclusion complex (c), and C. inophyllum resin/γ-CD inclusion complex (d).

Determination of MICs of 1/CDs  The MICs of 1 alone and 1 /CDs are listed in Table 3. Based on these results, the MICs of 1 in 1/CDs were calculated (Table 4). The inclusion complexes with CDs exhibit a potent antibacterial activity against S. aureus and E. coli at lower amounts of 1 than that of 1 alone (Tables 3 and 4) confirming that, the antibacterial activity of 1 is improved by the formation of the inclusion complexes with CDs. In particular, the 1/α-CD exhibited the most notable improvement in the antibacterial activity.

Table 3 MICs (μg/mL) of 1 and 1/CDs inclusion complex against Staphylococcus aureus and Escherichia coli.

	S. aureus	E. coli
1	32	16
1/α-CD inclusion complex	32	16
1/β-CD inclusion complex	64	16
1/γ-CD inclusion complex	64	16

Table 4 The calculated MICs of 1 (µg/mL) in 1/CDs inclusion complex.

	S. aureus	E. coli
1/α-CD inclusion complex	11	5.6
1/β-CD inclusion complex	20	5.1
1/γ-CD inclusion complex	18	4.6

Water solubility test via UV-VIS spectroscopy  As mentioned previously, C. inophyllum resin and 1 have low solubility in water. Therefore, we evaluated the effects of the inclusion with CDs on the water solubility by examining the UV-VIS absorption. The UV spectra of C. inophyllum resin/α-CD, C. inophyllum resin, and α-CD in water are shown in Fig. 3 (a), and the spectra of 1/α-CD, 1, and α-CD in water are shown in Fig. 3 (b). The aqueous solution of α-CD does not exhibit an absorption peak at 324 nm, which is the maximum absorption wavelength of C. inophyllum resin and 1. The absorption maxima at 324 nm for C. inophyllum resin/α-CD, C. inophyllum resin, 1/α-CD, and 1 are 0.43, 0.32, 0.65, and 0.35, respectively. Calculated the solubilities using ε of 324 nm in acetonitrile (9.4 × 10³), the concentrations of 1 were 37.3 for 1 alone and 69.3 µM for 1/α-CD.

Fig. 3.

UV spectra of aqueous solution: Calophyllum inophyllum resin/α-CD inclusion complex, C. inophyllum resin, and α-CD (a); 1/α-CD inclusion complex, 1, and α-CD (b).

Phase solubility study  This study was conducted to investigate the molar ratio at which 1 and α-CD form inclusion complexes. Excess amounts of 1 were added to several concentrations of α-CD, and the concentrations of 1 dissolved in each α-CD solution were measured and plotted. The solubility of 1 increase linearly with increasing α-CD concentration in aqueous medium (Fig. 4).

Fig. 4.

Phase solubility diagram of 1 with α-CD.

All values are given standard error for three measurements (n = 3).

¹H NMR analysis  ¹H NMR was used to investigated whether 1 and α-CD formed an inclusion complex. The chemical shifts of the H-3 and H-5 protons of CDs can indicate the formation of inclusion complexes (Mura et al., 2014). Therefore, we observed the ¹H NMR chemical shifts of Δδ_H-3 and Δδ_H-5 for α-CD. As listed in Table 5, the δ_H-3 and δ_H-5 of the α-CD inclusion complex shift upfield, by 0.0040 and 0.0024 ppm, respectively. The chemical shifts of H-4′ and H-5′ of 1 also shift upfield, by 0.0362 and 0.0262 ppm, respectively (Table 6).

Table 5 ¹H NMR chemical shift δ and Δδ of α-CD in free or inclusion complex.

α-CD	δ of α-CD	δ of 1/α-CD	Δδ
H-3	3.7796	3.7756	−0.0040
H-5	3.5891	3.5867	−0.0024

Table 6 ¹H NMR chemical shift δ and Δδ of 1 in free or inclusion complex.

1	δ of 1	δ of 1/α-CD	Δδ
H-4′	1.5226	1.4864	−0.0362
H-5′	1.4585	1.4323	−0.0262

Discussion

In this study, we attempted to improve the water solubility and antibacterial activity of C. inophyllum resin and 1. We therefore performed to form an inclusion complexes of C. inophyllum resin and 1 with CDs.

The CD inclusion complexes exhibit antibacterial activity against S. aureus and E. coli at lower amounts of the C. inophyllum resin or 1 than that of C. inophyllum resin or 1 alone (Tables 1–4). The improvement in the antibacterial activity was most notable for the α-CD inclusion complexes. C. inophyllum resin/α-CD exhibits antibacterial activity against S. aureus and E. coli at concentrations of 22 and 11 µg/mL, respectively (Table 2). However, C. inophyllum resin alone exhibits antibacterial activity against S. aureus and E. coli at MICs of 64 and 16 µg/mL, respectively (Table 1). Similarly, 1/α-CD exhibits antibacterial activity against S. aureus and E. coli at concentrations of 11 and 5.6 µg/mL, respectively (Table 4). In contrast, 1 alone exhibits antibacterial activity against S. aureus and E. coli at MICs of 32 and 16 µg/mL, respectively (Table 3). From the above results, the formation of inclusion complexes of C. inophyllum resin and 1 with CDs were confirmed to improve the antibacterial activity. In the water solubility test, the absorption maxima of the α-CD inclusion complex of C. inophyllum resin and 1 are 0.11 and 0.3, respectively (Fig. 3), at 324 nm. The solubility of 1 was confirmed to be 2-fold increased by CD inclusion effect. These results indicate that the formation of inclusion complex with α-CD enhances the water solubility of C. inophyllum resin and 1. The improved antibacterial activity of the inclusion complexes of CDs under aqueous conditions is possibly due to their improved water solubility.

From the results of the phase solubility study, the concentration of 1 increases linearly with increasing α-CD concentration in the aqueous medium (Fig. 4). This linearity is known as the AL-type characteristic, indicating that the stoichiometries of 1/α-CD are equal (Gao et al., 2020; Jiang et al., 2019; Higuchi et al., 1965) and that 1 and α-CD form an inclusion complex at a molar ratio of 1:1.

¹H NMR analysis was used to confirm the inclusion of guests molecules in the CD cavities: when a hydrophobic guest enters the CD cavity, the chemical shifts of the protons inside the CD (H-3 and H-5) and those of the inclusion guest move upfield (Kaur et al., 2019) (Fig. 5). The H-3 and H-5 of α-CD shift upfield in the inclusion complex (Table 5). This indicated that 1 was encapsulated in α-CD. The chemical shift differences in the CD inclusion complexes can indicate the level of inclusion: Δδ_H-3 < Δδ_H-5 indicates complete inclusion, whereas Δδ_H-3 > Δδ_H-5 indicates partial inclusion (Mura et al., 2014). In this study, Δδ_H-3 (0.0040) > Δδ_H-5 (0.0024) indicating that 1 was partially encapsulated in the α-CD cavity. In addition, an upfield shift of H-4′ and H-5′ of 1 is observed, suggesting that the liposouble prenyl group of 1 is partially encapsulated in the α-CD cavity (Francy et al., 2022) (Table 6). We also confirmed whether 1 and α-CD formed an inclusion complex using FT-IR spectra. Because 1 and α-CD interacted weakly, there was no difference in intensity and pattern of each IR spectrum (data not shown). This is supported by the ¹H NMR results (1 is partially included in α-CD).

Fig. 5.

Model of cyclodextrins.

Conclusions

This study focused on improving the water solubility and antibacterial activity of C. inophyllum resin and 1 by forming inclusion complexes with CDs. The water solubility and antibacterial activity of C. inophyllum resin and 1 improved through the formation of inclusion complexes with CDs. Among the analyzed samples, the inclusion complex with α-CD exhibited the strongest antibacterial activity. These results confirm the significant potential of C. inophyllum resin, and 1 eradicate foodborne pathogens.

Acknowledgements  We thank Professor Shuichi Masuda and Dr. Yuko Shimamura for their cooperation with the antibacterial assay. This research was supported by the JSPS KAKENHI grant JP20J23632.

Conflict of interest  There are no conflicts of interest to declare.

Abbreviations

HPLC

high-performance liquid chromatography

CD

cyclodextrin

MIC

minimal inhibitory concentration

NMR

nuclear magnetic resonance

TMS

tetramethylsilane

DMSO

dimethyl sulfoxide

FT-IR

Fourier transform infrared spectroscopy

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